Original Article 361
Authors
S. R. M. Ibrahim1, 2, G. A. Mohamed3, 4, M. F. Zayed1, 5, H. M. Sayed2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ acanthostrongylophora ingens ● ▶ pyrimidine-β-carboline ● ▶ pyrimidine-γ-carboline ● ▶ ingenine ● ▶ cytotoxic activity ●
Abstract As a continuation of the work on EtOAc fraction of the Indonesian sponge Acanthostrongylophora ingens, 2 new alkaloids: one pyrimidine-βcarboline alkaloid named ingenine A (2) and one pyrimidine-γ-carboline alkaloid named ingenine B (3), along with annomontine (1) were isolated. Their structures were unambiguously estab-
lished on the basis of NMR spectroscopy (1 H, 13C, 1 H-1 H COSY, HMQC, and HMBC) and mass spectral data. This is the first report of isolation pyrimidine-γ-carboline alkaloid from natural source. Compounds 1 and 3 showed pronounced cytotoxicity against the murine lymphoma L5178Y cancer cell line with ED50 7.8 and 9.1 μg/ mL respectively, while compound 2 showed weak activity.
Introduction
Materials and Methods
The genus Acanthostrongylophora (order Haplosclerida; family Petrosiidae) is known to be a rich source of manzamine-type alkaloids [1, 2]. Manzamine-related metabolites are known to exhibit anti-malarial, anti-leishmanial, anti-tuberculosis, cytotoxic, and anti-HIV activities [1, 2]. Also, they had significant inhibitory activity of GSK3, an enzyme implicated in Alzheimer’s disease pathology [2]. Previous phytochemical study of Indonesian sponge Acanthostrongylophora ingens led to isolation of a new β-carboline alkaloid; acanthomine A, together with annomontine and 1,2,3,4-tetrahydronorharman-1-one [3]. In our continuing research on pharmacologically active agents, we report herein the isolation and structural elucidation of 2 new alkaloids: a pyrimidine β-carboline alkaloid, ingenine A (2) and a pyrimidine-γ-carboline alkaloid, ingenine B (3), along with annomontine (1) ● ▶ Fig. 1. Their structures were verified by various spectroscopic methods. The isolated compounds were evaluated for their cytotoxic activity against L5178Y a murine lymphoma cancer cell line.
General experimental procedures
▼
▼ received 25.04.2014 accepted 30.06.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384577 Published online: July 22, 2014 Drug Res 2015; 65: 361–365 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. R. M. Ibrahim, Associate Professor of Pharmacognosy Department of Pharmacognosy and Pharmaceutical Chemistry College of Pharmacy Taibah University Postal: College of Pharmacy Taibah University Al-Madinah Al-munawarah 41477 Kingdom of Saudi Arabia P. O. box 30019 Tel.: + 966/581/183 034 Fax: + 966/484/75 027 [email protected]
▼
Optical rotations were measured on a PerkinElmer Model 341 LC polarimeter (Perkin-Elmer, Waltham, MA, USA). Melting points were carried out in Electrothermal 9 100 Digital Melting Point (Electrothermal Engineering, Southend-on-Sea, Essex, UK). IR spectra were measured on Schimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). 1H NMR and 13C NMR experiments were performed on Bruker Unity 500, 125 MHz spectrometer (Bruker BioSpin, Billerica, MA, USA). ESIMS spectra were obtained with a Thermofinnigan LCQ DECA mass spectrometer coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector (ThermoFinnigan, Waltham, MA, USA). HRESIMS spectra were measured on a LTQ Orbitrap (ThermoFinnigan, Bremen, Germany). HPLC separations were performed on an HPLC system consisting of a Lachrom-Merck Hitachi L-7100 pump and a L-7400 UV detector using a C-18 column (300 × 8 mm i. d., prefilled with Eurospher 100, Knauer, Berlin, Germany), a flow rate of 5.0 mL/min. Column chromatographic separations were performed on silica gel 60 (0.040– 0.063 mm) and sephadex LH-20 (0.25–0.1 mm, Merck, Darmstadt, Germany). TLC analyses were carried out on aluminum sheets pre-coated with silica gel 60 F254 (Merck, Darmstadt, Germany).
Ibrahim SRM et al. Ingenines A-B, New Carboline Alkaloids … Drug Res 2015; 65: 361–365
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Ingenines A and B, Two New Alkaloids from the Indonesian Sponge Acanthostrongylophora ingens
362 Original Article All solvents were distilled prior to use. Spectral grade solvents were utilized for chromatographic analysis.
over silica gel column (130 g × 50 cm × 2 cm) using CH2Cl2:MeOH gradient to get 7 subfractions. Subfractions 2, 3, and 5 were previously investigated by authors [3]. Subfraction 4 (48 mg) was subjected to semi-preparative HPLC to yield compounds 1 (yellow crystalline solid, 10.6 mg) and 2 (orange red amorphous powder, 5.8 mg). Subfraction 6 (31 mg) was chromatographed over sephadex LH20 using MeOH as an eluent to get impure 3. Further purification of the 3 was accomplished by semi-preparative HPLC to give 3 (yellow amorphous powder, 6.9 mg).
Extraction and isolation
The sponges were collected by scuba diving at depth of 4–5 m from Ujung Pandang Island in Indonesia in 1996. Freshly collected sponges were frozen immediately after collection and then freeze-dried. A voucher specimen has been deposited in the Zoological Museum, University of Amsterdam, under the registration no. ZMAPOR. 17795. The freeze-dried sponge (120.0 g) was extracted with MeOH (4 × 500 mL). The combined MeOH extract was concentrated under reduced pressure to yield a dark brown viscous residue (3.8 g). The latter was subjected to vacuum liquid chromatography (VLC) using n-hexane, CH2Cl2, EtOAc, and MeOH. The EtOAC fraction was chromatographed
8
5a 8a
4a
4
1a 1
3 2N
Annomontine (1): Yellow crystalline solid. m. p. 246–247 °C; [α]D25 − 34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.23), 289 (3.98), and 311 (4.10) nm; 1H and 13C NMR data (DMSO-d6, 500 and 125 MHz) are given in ● ▶ Table 1; ( + ) ESIMS m/z 262 [M + H] + . Ingenine A (2): Orange red amorphous powder. [α]D25 + 16 ° (c 0.2, CH3OH); UV (MeOH) λmax (log ε) 219 (4.27), 286 (3.99), and 311 (3.80) nm; IR (KBr) νmax 3 066, 2 989, 1 610, and 1 247 cm − 1; ▶ Table 1; NMR data ((DMSO-d6, 500 and 125 MHz) are given in ● HRESIMS m/z 262.1011 [M + H] + (calcd for C15H12N5, 262.1014).
N N H
N H 5' 4'
6` 3' N
N 1` 2'
Ingenine B (3): Yellow amorphous powder. [α]D25 − 56 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 226 (4.35), 293 (3.95), and 372 (3.63) nm; IR (KBr) νmax 3 056, 2 996, and 1 615 cm − 1; NMR data ▶ Table 3; HRESIMS ((DMSO-d6, 500 and 125 MHz) are given in ● m/z 262.1007 [M + H] + (calcd for C15H12N5, 261.1014).
N NH2
N
H2N
1
2
Cytotoxicity study
The cytotoxicity against L5178Y (murine lymphoma cells) was determined using the microculture tetrazolium assay (MTT) and compared to that of untreated controls. Exponentially growing cells were harvested, counted, and diluted appropriately. Of the cell suspension, 50 μL containing 3 750 cells were pipetted into 96-well microtiter plates. Subsequently, 50 μL of a solution of the tested samples (10 µg/mL) was added to each well. The test plates were incubated at 37 °C with 5 % CO2 for 72 h. The media with all remaining tested sponge products are removed and the cells are subjected to washing several times to eliminate any remaining sponge product. A solution of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was prepared at 5 mg/mL in phosphate buffered saline (PBS; 1.5 mM KH2PO4,
N
N H N
N
NH2
3 Fig. 1 Structures of compounds 1–3 from the sponge Acanthostrongylophora ingens.
Position NH 1 3 4 4a 5a 5 6 7 8 8a 1a 2′ 4′ 5′ 6′
2
1
δH mult. (J in Hz)
δC
HMBC
δH mult. (J in Hz)
δC
11.90 s – 8.58 d (5.0) 8.43 d (5.0) – – 8.35 d (7.9) 7.35 t (7.9) 7.67 t (7.9) 7.79 d (7.9) – – – 8.49 d (5.3) 7.84 d (5.3) –
– 134.1 137.9 118.2 131.1 120.1 122.2 120.3 129.3 112.4 141.1 134.8 159.8 153.4 105.6 166.9
4a, 5a, 8a – 1, 4, 4a 1a, 5a – – 8a 5a, 8 5, 8a 6 – – – 1, 2′, 5′, 6′ 1, 4′ –
11.54 s – 8.51 d (5.0) 8.29 d (5.0) – – 8.31 d (7.9) 7.29 dt (7.9, 0.9) 7.64 dt (7.9, 0.9) 7.77 d (7.9) – – – 8.44 d (5.3) 7.68 d (5.3) –
– 135.8 137.6 116.7 130.0 119.8 121.7 119.5 128.6 112.0 140.4 134.0 163.9 158.7 105.3 163.9
Ibrahim SRM et al. Ingenines A-B, New Carboline Alkaloids … Drug Res 2015; 65: 361–365
Table 1 NMR spectroscopic data of compounds 1 and 2 (DMSO-d6, 500 and 125 MHz).
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6 5 7
Physical and spectroscopic data of compounds
Original Article 363
Results and Discussion
▼
Compound 2 was obtained as an orange red amorphous powder. It had an [α]D value of + 16 ° (c 0.2, CH3OH). The UV spectrum showed absorption bands at 219, 286, and 311 nm. Characteristic absorption bands at 3 066, 2 989, 1610, and 1 247 cm − 1 were observed in IR spectrum. The HRESIMS spectrum of 2 showed a pseudo-molecular ion peak at m/z 262.1011 [M + H] + . Combined with 1H and 13C NMR spectral data of 2, its molecular formula was determined as C15H11N5. NMR spectral data of 2 and annomontine (1) were quite similar ● ▶ Table 1. However, significant differences in the 1H and 13C NMR chemical shifts of 2-aminopyrimidine moiety, [α]D values [ + 16 ° for 2 and – 34 ° for 1], and retention times between 2 and 1 were observed. The 13C NMR and HMQC spectra showed the presence of 15 carbon signals: 8 methines and 7 quaternary carbons. The inspection of 1H NMR and 1H-1H COSY spectra showed 4 coupled signals at δH 8.35 (d, J = 7.9 Hz, H-5), 7.35 (t, J = 7.9 Hz, H-6), 7.67 (t, J = 7.9 Hz, H-7), and 7.79 (d, J = 7.9 Hz, H-8), which were assigned to indole moiety [3, 6, 7]. 2 deshielded doublets at δH 8.58 (d, J = 5.0 Hz, H-3) and 8.43 (d, J = 5.0 Hz, H-4) for pyridine moiety were observed. The presence of the indole and pyridine moieties was confirmed by the observed HMBC cross peaks ● ▶ Fig. 2. An exchangeable proton for the NH group at δH 11.90 (indolic NH) was also evident, which was 0.36 ppm downfield-shifted in comparison with this in 1 (δH 11.54). The attachment of the indole moiety
with pyridine ring to give a β-carboline moiety was established by the HMBC correlations of NH group to the carbon signals resonating at δC 131.1 (C-4a), 120.1 (C-5a), and 141.1 (C-8a) ▶ Fig. 2 [8]. An additional spin system for the 2-aminopyrimi● dine moiety at δH 8.49 (d, J = 5.3 Hz, H-4′) and 7.84 (d, J = 5.3 Hz, H-5′) was present. This was established by the 13C NMR signals at δC 159.8 (s, C-2′), 153.4 (d, C-4′), 105.6 (d, C-5′), and 166.9 (s, C-6′) ● ▶ Table 1. The attachment of the 2-aminopyrimidine moiety at C-1 of indole moiety was established by the HMBC correlation of H-5′ and H-4′ to C-1. From the above evidences, Compound 2 was a conformational isomer of 1, which differs in the orientation of the aminopyrimidine ring at C-1.
Conformational analysis
The conformational analysis of the 4-(9H-pyrido[3, 4-b]indol1-yl)pyrimidin-2-amine by using the computerized molecular modeling program SYBYL-X 2.0 (Tripos international, St. Louis, USA) revealed that, there are 2 conformational isomers resulted from the rotation of 2-methylpridine around the (C-C) single bond ● ▶ Fig. 3. This bond represents an axis of rotation for the 2-methylpyridine. When the potential energy was measured (E) for each isomer, it found that they have different energy values as explained in ● ▶ Table 2. The energy difference (ΔE) = 40.425– 34.632 = 5.793 KJ/mol. This difference between the 2 isomers allowed their separation by HPLC. The electrostatic potential map of each isomer ● ▶ Fig. 3 explains also the difference between the 2 isomers. Accordingly, the structure of 2 was unambiguously elucidated and named ingenine A. From the obtained data in ● ▶ Table 1, it was noticed that ingenine A is more energetically stable than annomontine (1). Compound 3 was isolated as yellow amorphous powder, with the molecular formula C15H11N5, determined by the HRESIMS pseudo-molecular ion peak at m/z 262.1007 [M + H] + . It had UV absorbances at 226, 293, and 372 nm. The IR spectrum showed absorption bands at 3 056, 2 996, and 1 615 cm − 1. The 1H NMR
Ingenine A (2)
N N N H
N H
N
N
COSY H 2N
N
2
HMBC
Fig. 2 Key 1 H-1H COSY and HMBC correlations of 2 and 3.
N
3
NH2
Annomontine (1) Fig. 3 Ingenine A (2) and its Electrostatic map on the upper part while, annomontine (1) and its electrostatic map on the lower part.
Ibrahim SRM et al. Ingenines A-B, New Carboline Alkaloids … Drug Res 2015; 65: 361–365
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6.5 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl; pH 7.4) and from this solution, 20 μL was pipetted into each well. The yellow MTT penetrates the healthy living cells and in the presence of mitochondrial dehydrogenases, MTT is transformed to its blue formazan complex. After an incubation period of 4 h at 37 °C in a humidified incubator with 5 % CO2, the medium was centrifuged (15 min, 20 °C, 210 × g) with 200 μL DMSO, the cells were lysed to liberate the formed formazan product. After thorough mixing, the absorbance was measured at 520 nm using a scanning microtiter-well spectrophotometer. The colour intensity is correlated with the number of healthy living cells. All experiments were carried out in triplicates and repeated 3 times. As negative controls, media with 0.1 % (v/v) EtOH were included in all experiments. A negative control was carried out by adding 50 μL of each the tested samples solutions (10 µg/mL) to 20 μL of the yellow MTT solution. The solution was allowed to stand at 37 °C for 4 h. There is no change in the yellow colour of MTT solution was occurred. The depsipeptide kahalalide F was used as a positive control [4, 5].
364 Original Article
Iso-
Total
mer
energy
1 2
40.425 34.632
Stretch
angle
bending
Van der
electric
Walla 3.803 3.517
5.429 5.136
– 0.41 – 0.44
39.154 39.429
– 7.551 – 13.01
Table 3 NMR spectroscopic data of compounds 3 (DMSO-d6, 500 and 125 MHz). Position
δH mult. (J in Hz)
δC
HMBC
NH 1 2 4 4a 5a 5 6 7 8 8a 1a 2′ 4′ 5′ 6′ NH2
12.30 s
– 140.2 128.3 100.6 129.8 124.1 126.7 129.7 123.2 126.0 141.3 127.0 163.9 159.3 106.1 163.5 –
1a, 4a, 5a
7.78 d (1.8) 7.25 d (1.8) – – 7.64 dd (6.0, 2.5) 7.62 ddd (8.2, 6.0, 2.5) 8.36 ddd (8.2, 6.0, 2.5) 8.12 dd (6.0, 2.5) – – – 8.49 d (5.0) 7.82 d (5.0) – 7.05 brs
4, 1a, 4a 2, 1a, 4a – – 6, 7, 4a, 5a, 8a 8, 5a, 8a 6, 8, 5a, 8a 6, 5a – – – 1, 2′, 6′, 5′ 1, 4′, 6′ – –
Table 4 Cytotoxicity of the isolated carbolines against L5178Y cell line. Compound
% Growth inhibition L5178Y
ED50 µg/mL
10 µg/mL Annonomontine (1) Ingenine A (2) Ingenine B (3) Kahalalide F
100 12.5 83.5
7.8
Authors
S. R. M. Ibrahim1, 2, G. A. Mohamed3, 4, M. F. Zayed1, 5, H. M. Sayed2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ acanthostrongylophora ingens ● ▶ pyrimidine-β-carboline ● ▶ pyrimidine-γ-carboline ● ▶ ingenine ● ▶ cytotoxic activity ●
Abstract As a continuation of the work on EtOAc fraction of the Indonesian sponge Acanthostrongylophora ingens, 2 new alkaloids: one pyrimidine-βcarboline alkaloid named ingenine A (2) and one pyrimidine-γ-carboline alkaloid named ingenine B (3), along with annomontine (1) were isolated. Their structures were unambiguously estab-
lished on the basis of NMR spectroscopy (1 H, 13C, 1 H-1 H COSY, HMQC, and HMBC) and mass spectral data. This is the first report of isolation pyrimidine-γ-carboline alkaloid from natural source. Compounds 1 and 3 showed pronounced cytotoxicity against the murine lymphoma L5178Y cancer cell line with ED50 7.8 and 9.1 μg/ mL respectively, while compound 2 showed weak activity.
Introduction
Materials and Methods
The genus Acanthostrongylophora (order Haplosclerida; family Petrosiidae) is known to be a rich source of manzamine-type alkaloids [1, 2]. Manzamine-related metabolites are known to exhibit anti-malarial, anti-leishmanial, anti-tuberculosis, cytotoxic, and anti-HIV activities [1, 2]. Also, they had significant inhibitory activity of GSK3, an enzyme implicated in Alzheimer’s disease pathology [2]. Previous phytochemical study of Indonesian sponge Acanthostrongylophora ingens led to isolation of a new β-carboline alkaloid; acanthomine A, together with annomontine and 1,2,3,4-tetrahydronorharman-1-one [3]. In our continuing research on pharmacologically active agents, we report herein the isolation and structural elucidation of 2 new alkaloids: a pyrimidine β-carboline alkaloid, ingenine A (2) and a pyrimidine-γ-carboline alkaloid, ingenine B (3), along with annomontine (1) ● ▶ Fig. 1. Their structures were verified by various spectroscopic methods. The isolated compounds were evaluated for their cytotoxic activity against L5178Y a murine lymphoma cancer cell line.
General experimental procedures
▼
▼ received 25.04.2014 accepted 30.06.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384577 Published online: July 22, 2014 Drug Res 2015; 65: 361–365 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. R. M. Ibrahim, Associate Professor of Pharmacognosy Department of Pharmacognosy and Pharmaceutical Chemistry College of Pharmacy Taibah University Postal: College of Pharmacy Taibah University Al-Madinah Al-munawarah 41477 Kingdom of Saudi Arabia P. O. box 30019 Tel.: + 966/581/183 034 Fax: + 966/484/75 027 [email protected]
▼
Optical rotations were measured on a PerkinElmer Model 341 LC polarimeter (Perkin-Elmer, Waltham, MA, USA). Melting points were carried out in Electrothermal 9 100 Digital Melting Point (Electrothermal Engineering, Southend-on-Sea, Essex, UK). IR spectra were measured on Schimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). 1H NMR and 13C NMR experiments were performed on Bruker Unity 500, 125 MHz spectrometer (Bruker BioSpin, Billerica, MA, USA). ESIMS spectra were obtained with a Thermofinnigan LCQ DECA mass spectrometer coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector (ThermoFinnigan, Waltham, MA, USA). HRESIMS spectra were measured on a LTQ Orbitrap (ThermoFinnigan, Bremen, Germany). HPLC separations were performed on an HPLC system consisting of a Lachrom-Merck Hitachi L-7100 pump and a L-7400 UV detector using a C-18 column (300 × 8 mm i. d., prefilled with Eurospher 100, Knauer, Berlin, Germany), a flow rate of 5.0 mL/min. Column chromatographic separations were performed on silica gel 60 (0.040– 0.063 mm) and sephadex LH-20 (0.25–0.1 mm, Merck, Darmstadt, Germany). TLC analyses were carried out on aluminum sheets pre-coated with silica gel 60 F254 (Merck, Darmstadt, Germany).
Ibrahim SRM et al. Ingenines A-B, New Carboline Alkaloids … Drug Res 2015; 65: 361–365
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Ingenines A and B, Two New Alkaloids from the Indonesian Sponge Acanthostrongylophora ingens
362 Original Article All solvents were distilled prior to use. Spectral grade solvents were utilized for chromatographic analysis.
over silica gel column (130 g × 50 cm × 2 cm) using CH2Cl2:MeOH gradient to get 7 subfractions. Subfractions 2, 3, and 5 were previously investigated by authors [3]. Subfraction 4 (48 mg) was subjected to semi-preparative HPLC to yield compounds 1 (yellow crystalline solid, 10.6 mg) and 2 (orange red amorphous powder, 5.8 mg). Subfraction 6 (31 mg) was chromatographed over sephadex LH20 using MeOH as an eluent to get impure 3. Further purification of the 3 was accomplished by semi-preparative HPLC to give 3 (yellow amorphous powder, 6.9 mg).
Extraction and isolation
The sponges were collected by scuba diving at depth of 4–5 m from Ujung Pandang Island in Indonesia in 1996. Freshly collected sponges were frozen immediately after collection and then freeze-dried. A voucher specimen has been deposited in the Zoological Museum, University of Amsterdam, under the registration no. ZMAPOR. 17795. The freeze-dried sponge (120.0 g) was extracted with MeOH (4 × 500 mL). The combined MeOH extract was concentrated under reduced pressure to yield a dark brown viscous residue (3.8 g). The latter was subjected to vacuum liquid chromatography (VLC) using n-hexane, CH2Cl2, EtOAc, and MeOH. The EtOAC fraction was chromatographed
8
5a 8a
4a
4
1a 1
3 2N
Annomontine (1): Yellow crystalline solid. m. p. 246–247 °C; [α]D25 − 34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.23), 289 (3.98), and 311 (4.10) nm; 1H and 13C NMR data (DMSO-d6, 500 and 125 MHz) are given in ● ▶ Table 1; ( + ) ESIMS m/z 262 [M + H] + . Ingenine A (2): Orange red amorphous powder. [α]D25 + 16 ° (c 0.2, CH3OH); UV (MeOH) λmax (log ε) 219 (4.27), 286 (3.99), and 311 (3.80) nm; IR (KBr) νmax 3 066, 2 989, 1 610, and 1 247 cm − 1; ▶ Table 1; NMR data ((DMSO-d6, 500 and 125 MHz) are given in ● HRESIMS m/z 262.1011 [M + H] + (calcd for C15H12N5, 262.1014).
N N H
N H 5' 4'
6` 3' N
N 1` 2'
Ingenine B (3): Yellow amorphous powder. [α]D25 − 56 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 226 (4.35), 293 (3.95), and 372 (3.63) nm; IR (KBr) νmax 3 056, 2 996, and 1 615 cm − 1; NMR data ▶ Table 3; HRESIMS ((DMSO-d6, 500 and 125 MHz) are given in ● m/z 262.1007 [M + H] + (calcd for C15H12N5, 261.1014).
N NH2
N
H2N
1
2
Cytotoxicity study
The cytotoxicity against L5178Y (murine lymphoma cells) was determined using the microculture tetrazolium assay (MTT) and compared to that of untreated controls. Exponentially growing cells were harvested, counted, and diluted appropriately. Of the cell suspension, 50 μL containing 3 750 cells were pipetted into 96-well microtiter plates. Subsequently, 50 μL of a solution of the tested samples (10 µg/mL) was added to each well. The test plates were incubated at 37 °C with 5 % CO2 for 72 h. The media with all remaining tested sponge products are removed and the cells are subjected to washing several times to eliminate any remaining sponge product. A solution of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) was prepared at 5 mg/mL in phosphate buffered saline (PBS; 1.5 mM KH2PO4,
N
N H N
N
NH2
3 Fig. 1 Structures of compounds 1–3 from the sponge Acanthostrongylophora ingens.
Position NH 1 3 4 4a 5a 5 6 7 8 8a 1a 2′ 4′ 5′ 6′
2
1
δH mult. (J in Hz)
δC
HMBC
δH mult. (J in Hz)
δC
11.90 s – 8.58 d (5.0) 8.43 d (5.0) – – 8.35 d (7.9) 7.35 t (7.9) 7.67 t (7.9) 7.79 d (7.9) – – – 8.49 d (5.3) 7.84 d (5.3) –
– 134.1 137.9 118.2 131.1 120.1 122.2 120.3 129.3 112.4 141.1 134.8 159.8 153.4 105.6 166.9
4a, 5a, 8a – 1, 4, 4a 1a, 5a – – 8a 5a, 8 5, 8a 6 – – – 1, 2′, 5′, 6′ 1, 4′ –
11.54 s – 8.51 d (5.0) 8.29 d (5.0) – – 8.31 d (7.9) 7.29 dt (7.9, 0.9) 7.64 dt (7.9, 0.9) 7.77 d (7.9) – – – 8.44 d (5.3) 7.68 d (5.3) –
– 135.8 137.6 116.7 130.0 119.8 121.7 119.5 128.6 112.0 140.4 134.0 163.9 158.7 105.3 163.9
Ibrahim SRM et al. Ingenines A-B, New Carboline Alkaloids … Drug Res 2015; 65: 361–365
Table 1 NMR spectroscopic data of compounds 1 and 2 (DMSO-d6, 500 and 125 MHz).
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6 5 7
Physical and spectroscopic data of compounds
Original Article 363
Results and Discussion
▼
Compound 2 was obtained as an orange red amorphous powder. It had an [α]D value of + 16 ° (c 0.2, CH3OH). The UV spectrum showed absorption bands at 219, 286, and 311 nm. Characteristic absorption bands at 3 066, 2 989, 1610, and 1 247 cm − 1 were observed in IR spectrum. The HRESIMS spectrum of 2 showed a pseudo-molecular ion peak at m/z 262.1011 [M + H] + . Combined with 1H and 13C NMR spectral data of 2, its molecular formula was determined as C15H11N5. NMR spectral data of 2 and annomontine (1) were quite similar ● ▶ Table 1. However, significant differences in the 1H and 13C NMR chemical shifts of 2-aminopyrimidine moiety, [α]D values [ + 16 ° for 2 and – 34 ° for 1], and retention times between 2 and 1 were observed. The 13C NMR and HMQC spectra showed the presence of 15 carbon signals: 8 methines and 7 quaternary carbons. The inspection of 1H NMR and 1H-1H COSY spectra showed 4 coupled signals at δH 8.35 (d, J = 7.9 Hz, H-5), 7.35 (t, J = 7.9 Hz, H-6), 7.67 (t, J = 7.9 Hz, H-7), and 7.79 (d, J = 7.9 Hz, H-8), which were assigned to indole moiety [3, 6, 7]. 2 deshielded doublets at δH 8.58 (d, J = 5.0 Hz, H-3) and 8.43 (d, J = 5.0 Hz, H-4) for pyridine moiety were observed. The presence of the indole and pyridine moieties was confirmed by the observed HMBC cross peaks ● ▶ Fig. 2. An exchangeable proton for the NH group at δH 11.90 (indolic NH) was also evident, which was 0.36 ppm downfield-shifted in comparison with this in 1 (δH 11.54). The attachment of the indole moiety
with pyridine ring to give a β-carboline moiety was established by the HMBC correlations of NH group to the carbon signals resonating at δC 131.1 (C-4a), 120.1 (C-5a), and 141.1 (C-8a) ▶ Fig. 2 [8]. An additional spin system for the 2-aminopyrimi● dine moiety at δH 8.49 (d, J = 5.3 Hz, H-4′) and 7.84 (d, J = 5.3 Hz, H-5′) was present. This was established by the 13C NMR signals at δC 159.8 (s, C-2′), 153.4 (d, C-4′), 105.6 (d, C-5′), and 166.9 (s, C-6′) ● ▶ Table 1. The attachment of the 2-aminopyrimidine moiety at C-1 of indole moiety was established by the HMBC correlation of H-5′ and H-4′ to C-1. From the above evidences, Compound 2 was a conformational isomer of 1, which differs in the orientation of the aminopyrimidine ring at C-1.
Conformational analysis
The conformational analysis of the 4-(9H-pyrido[3, 4-b]indol1-yl)pyrimidin-2-amine by using the computerized molecular modeling program SYBYL-X 2.0 (Tripos international, St. Louis, USA) revealed that, there are 2 conformational isomers resulted from the rotation of 2-methylpridine around the (C-C) single bond ● ▶ Fig. 3. This bond represents an axis of rotation for the 2-methylpyridine. When the potential energy was measured (E) for each isomer, it found that they have different energy values as explained in ● ▶ Table 2. The energy difference (ΔE) = 40.425– 34.632 = 5.793 KJ/mol. This difference between the 2 isomers allowed their separation by HPLC. The electrostatic potential map of each isomer ● ▶ Fig. 3 explains also the difference between the 2 isomers. Accordingly, the structure of 2 was unambiguously elucidated and named ingenine A. From the obtained data in ● ▶ Table 1, it was noticed that ingenine A is more energetically stable than annomontine (1). Compound 3 was isolated as yellow amorphous powder, with the molecular formula C15H11N5, determined by the HRESIMS pseudo-molecular ion peak at m/z 262.1007 [M + H] + . It had UV absorbances at 226, 293, and 372 nm. The IR spectrum showed absorption bands at 3 056, 2 996, and 1 615 cm − 1. The 1H NMR
Ingenine A (2)
N N N H
N H
N
N
COSY H 2N
N
2
HMBC
Fig. 2 Key 1 H-1H COSY and HMBC correlations of 2 and 3.
N
3
NH2
Annomontine (1) Fig. 3 Ingenine A (2) and its Electrostatic map on the upper part while, annomontine (1) and its electrostatic map on the lower part.
Ibrahim SRM et al. Ingenines A-B, New Carboline Alkaloids … Drug Res 2015; 65: 361–365
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6.5 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl; pH 7.4) and from this solution, 20 μL was pipetted into each well. The yellow MTT penetrates the healthy living cells and in the presence of mitochondrial dehydrogenases, MTT is transformed to its blue formazan complex. After an incubation period of 4 h at 37 °C in a humidified incubator with 5 % CO2, the medium was centrifuged (15 min, 20 °C, 210 × g) with 200 μL DMSO, the cells were lysed to liberate the formed formazan product. After thorough mixing, the absorbance was measured at 520 nm using a scanning microtiter-well spectrophotometer. The colour intensity is correlated with the number of healthy living cells. All experiments were carried out in triplicates and repeated 3 times. As negative controls, media with 0.1 % (v/v) EtOH were included in all experiments. A negative control was carried out by adding 50 μL of each the tested samples solutions (10 µg/mL) to 20 μL of the yellow MTT solution. The solution was allowed to stand at 37 °C for 4 h. There is no change in the yellow colour of MTT solution was occurred. The depsipeptide kahalalide F was used as a positive control [4, 5].
364 Original Article
Iso-
Total
mer
energy
1 2
40.425 34.632
Stretch
angle
bending
Van der
electric
Walla 3.803 3.517
5.429 5.136
– 0.41 – 0.44
39.154 39.429
– 7.551 – 13.01
Table 3 NMR spectroscopic data of compounds 3 (DMSO-d6, 500 and 125 MHz). Position
δH mult. (J in Hz)
δC
HMBC
NH 1 2 4 4a 5a 5 6 7 8 8a 1a 2′ 4′ 5′ 6′ NH2
12.30 s
– 140.2 128.3 100.6 129.8 124.1 126.7 129.7 123.2 126.0 141.3 127.0 163.9 159.3 106.1 163.5 –
1a, 4a, 5a
7.78 d (1.8) 7.25 d (1.8) – – 7.64 dd (6.0, 2.5) 7.62 ddd (8.2, 6.0, 2.5) 8.36 ddd (8.2, 6.0, 2.5) 8.12 dd (6.0, 2.5) – – – 8.49 d (5.0) 7.82 d (5.0) – 7.05 brs
4, 1a, 4a 2, 1a, 4a – – 6, 7, 4a, 5a, 8a 8, 5a, 8a 6, 8, 5a, 8a 6, 5a – – – 1, 2′, 6′, 5′ 1, 4′, 6′ – –
Table 4 Cytotoxicity of the isolated carbolines against L5178Y cell line. Compound
% Growth inhibition L5178Y
ED50 µg/mL
10 µg/mL Annonomontine (1) Ingenine A (2) Ingenine B (3) Kahalalide F
100 12.5 83.5
7.8
