Abstract
Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinol isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g. Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3β (GSK-3β), which is a putative target of manzamines. Based on the results presented here it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.
Introduction
Marine invertebrates have been recognized as an important source of pharmacologically unique bioactive natural products. Apart from human medicines, the research involving marine natural products in the last three decades has also generated significant discoveries that are now utilized routinely as pharmacological tools with unique cellular targets. Some of them have become indispensable tools in biochemical research and played significant roles in the recent advancements of life sciences. In our search for bioactive compounds against infectious and neurological diseases, we have focused on marine alkaloids isolated from sponges and in particular the manzamine alkaloids.
Manzamines are characterized by a unique 5-, 6-, 6-, 8-, 13-membered heterocyclic ring system coupled to a β-carboline moiety. The first representative of this class, manzamine A (1), was isolated from the marine sponge Haliclona sp. collected near Manzamo Island by Higa and coworkers in 1986 and its structure including absolute configuration was established by X-ray diffraction.1 In recent years, the manzamines have been regarded as an interesting group of marine alkaloids with extraordinary biological activities, and as a result the molecules have received considerable attention for their chemistry and pharmacology. To date, over 80 manzamine related alkaloids have been isolated from more than 16 species of marine sponges belonging to 5 families distributed from the Red Sea to Indonesia, which suggested a potential microbial origin of manzamine alkaloids.2,3 The manzamine alkaloids have shown a variety of bioactivities including antimicrobial,4,5,6,7, antiparasitic,8 cytotoxic,1,9 antineuroinflammatory10,11,12,13 and pesticidal.14
The greatest potential for the manzamine alkaloids appears to be against malaria and neuroinflammation which is an interesting combination and may make it possible to treat both the infection and the symptoms with a single drug. The most effective and widely used antimalarial drugs are those derived from quinine and artemisinin, which are natural products from terrestrial plants. Our previous studies showed that the marine natural products 1 and 8-hydroxymanzamine A (2) exhibited improved potency against the malarial parasites both in vitro and in vivo relative to chloroquine and artemisinin.15 A remarkable aspect of 1 is its ability to prolong the survival of highly parasitemic mice with significant recovery after a single injection. Oral administration of 1 (2×100 μmol/Kg) and 2 (2×100 μmol/Kg) produced a significant reduction (90%) in parasitemia. All mice treated with a single dose (50 or 100 μmol/kg) of 1 or 2 showed significant improvements in survival times over mice treated with chloroquine or artemisinin.16 Significant variation of antimalarial activity among naturally isolated manzamines such as manzamine F (14) and ircinal A (19) provided some interesting information regarding the structure-activity relationship of these alkaloids.4, 7,15, 16, 17, 12
Manzamines have also been reported to have antimicrobial activities against a number of bacteria and fungi.3 In particular most naturally isolated manzamines were active against Mycobacterium tuberculosis.15,16,18 Compounds 1 and 2 showed 98-99% inhibition of Mycobacterium tuberculosis with MIC endpoints of 1.56 and 0.91 μg/mL, respectively.7 1 showed antimicrobial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus 7,17,12 with IC50 values of 0.5 and 0.7 μg/mL, respectively. 19
Several manzamines were reported to modulate the generation of O2- and TXB2 generated by activated rat neonatal microglia.12,13 1 was the most potent inhibitor and did not show in vitro toxicity to microglia, suggesting that it is a good candidate for treatment of neuroinflammatory diseases.10,11
Recently, 1 and related derivatives have been identified as a new class of GSK-3β inhibitors.20 Additionally, 1 was shown to be effective in decreasing tau hyper-phosphorylation in human neuroblastoma cell lines, a demonstration of its ability to enter cells and to interfere with tau pathology. Inhibition studies of 1 against five different kinases related to GSK-3β including CDK-1, PKA, CDK-5, MAPK and GSK-3α showed that manzamine A specifically inhibited GSK-3β and CDK-5,20 the two kinases involved in tau pathological hyperphosphorylation.21 These results suggest that the manzamines may represent a new class from which more potent and selective GSK-3/CDK5 inhibitors could be designed as potential therapeutic agents for Alzheimer's disease. In addition these results suggest that a group of closely related kinases may be responsible for the diverse activity seen for this class and that targeting Mycobacterium tuberculosis (Mtb) and plasmodial kinases may indeed be a viable approach to drug discovery and development for Mtb and malaria.
The unique structure and extraordinary bioactivity of manzamine alkaloids has attracted considerable interest from synthetic chemists due to a challenging structure for total synthesis.22,23,24 Methodology studies towards the synthesis of manzamine structural units have also been reported25,26,27,28 and a group of simplified analogues of 1 and manzamine C were synthesized.29,30,31 The simplified manzamine C products showed equal or more potent cytotoxicity than the natural manzamine C, while simplified 1 products showed significantly reduced antimalarial activity.
Modification of 1 using olefin metathesis has been carried out to generate novel structures with important biological properties.32 To better understand the structure-activity relationship and to generate better leads as potential drug candidates, we synthesized a number of new analogues of manzamines and evaluated their biological activity. An evaluation of the activity was made using molecular docking to an ATP-noncompetitive binding site of human GSK-3β.
Results and Discussion
Chemistry
To explore the structure-activity relationship, diversity is the first thought. Modifications were carried out on different positions and functional groups of manzamines to generate diverse analogues. For this reason four major manzamine alkaloids, 1, 2, 14, and 19, were chosen as starting material for the synthesis of analogues, although 14 and 19 only show marginal biological activity. Large quantities of these alkaloids had been produced from a common Indonesian sponge Acanthostrongylophora sp. through an optimized isolation procedure. Due to the number of potential reactive functional groups and instability of large and complex molecules such as manzamines, only mild and highly efficient reactions were employed for modification of manzamines. Thirty-nine analogues were synthesized in total, using various synthetic strategies, including sixteen analogues of 1, four of 2, eleven of 14, and eight of 19.
N-Alkylations (Scheme 1) (6a-i) of the β-carboline moiety with various alkyl halides were carried out starting with 1 in the presence of sodium hydride in dimethyl formamide at 0 °C. The alkylated products were obtained in good yield (81-87%). One and two dimensional NMR showed that alkylation occurred at the 9-N position.
Scheme 1.
Modification of manzamine A.
Reductions of 1 and 2 were performed in the presence of hydrogen-10% Pd/C in ethanol at room temperature for 4h. Manzamine A afforded a major product, 15,16,32,33-tetrahydromanzamine A (3) and a minor product 5, and 2 gave a major product 4 and a minor product 12.
1 was nitrated by sodium nitrate using TFA as a solvent to yield two nitrated products 7 and 8. The 6-nitromanzamine A (8) was generated in excess when compared to 8-nitromanzamine (7). These two products could be readily purified by flash column chromatography. Refluxing of 1 and 2 with acetic anhydride and potassium carbonate in dry acetone resulted in dehydration product 10 and 11. Oxidation of 1 with m-chloroperoxybenzoic acid in the presence of a catalytic amount of sodium bicarbonate in dichloromethane (DCM) solvent gave the corresponding N-oxides (13) without disturbing the double bond.
Modification of 14 was focused on the carbonyl group in the bottom eight-membered ring (Scheme 2). The carbonyl functional group was first reduced when treated with sodium borohydride to give a mixture of hydroxylated products and only one isomer (16) was obtained after purification. 14 was treated with Grignard reagent in dry THF to give the 31-alkyl product (18). Manzamine F-31-hydrazone (15) was obtained by heating 14 with hydrazine hydrate in ethanol. Knovenagel condensation of 14 with different aromatic aldehydes in the presence of a catalytic amount of piperidine yielded α, β-unsaturated products (17a-h).
Scheme 2.
Modifications beginning with manzamine F.
We next focused our efforts on converting 19 (Scheme 3) to analogues of 1 with a modified β-carboline moiety, which were otherwise difficult to synthesize directly from 1. 19 was reacted with substituted tryptamines in the presence of catalytic amounts of trifluoroacetic acid and activated molecular sieves in DCM to yield products through Pictet-Spengler cyclization. The Pictet-Spengler products33 were further aromatized with DDQ to yield substituted analogues of 1 (21a-d).
Scheme 3.
Modifications beginning with ircinal.
All of the above analogues were characterized by analysis of spectral data, provided in the experimental section and were evaluated for antimalarial, antimicrobial and anti-inflammatory activities.
Biological Activity
In vitro Antimalarial activity
All analogues were evaluated in vitro for antiprotozoal activity against Plasmodium falciparum (D6 and W2 clones). In addition they were also tested for cytotoxicity against mammalian kidney fibroblasts (Vero cells) as shown in Table 1. The activity profile exhibited varied results for each analogue against Plasmodium strains. 1 still provided the highest activity with IC50 of 4.5 ng/mL (D6 clone) and 8.0 ng/mL (W2 clone), which was comparable to the standard drugs chloroquine and artemisinin. Although toxicity to Vero cells was observed for 1 (IC50 200 ng/mL) but the selectivity index (ratio of IC50 for cytotoxicity to IC50 for antimalarial activity) for antimalarial activity was high (44 and 25 for D6 and W2 clones, respectively). Introducing an alkyl substituent on 9-N (6a-i) decreased the antimalarial activity significantly in both D6 and W2 clones showing that free 9-NH is required for antimalarial activity. Introducing lipophilic groups at 9N also diminished the antimalarial activity.
Table 1.
In vitro antimalarial activity against Plasmodium falciparum and cytotoxicity to mammalian cells*
| Compounds |
P. falciparum (D6 clone) |
P. falciparum (W2 clone) |
Cytotoxicity (Vero) |
|---|---|---|---|
| IC50 (ng/mL) | IC50 (ng/mL) | IC50 (μg/mL) | |
| 1 | 4.5 | 8.0 | 0.2 |
| 2 | 6.0 | 8.0 | 1.1 |
| 21b | 11 | 15 | 0.2 |
| 21a | 28 | 58 | 0.5 |
| 15 | 29 | 38 | NC |
| 18 | 77 | 86 | NC |
| 13 | 17 | 67 | NC |
| 8 | 18 | 28 | 270 |
| Chloroquine | 16 | 155 | NC |
| Artemisinin | 13 | 8 | NC |
For a full list of compounds and activity data, please see supporting material. NC = no cytotoxicity up to 4.76 μg/ml
Substitution on other positions of the β-carboline moiety also decreased antimalarial activity but not as much as did the substitution at 9-NH. The hydroxyl group at position 8 (2) did not produce a significant change in antimalarial activity (IC50 6.0 ng/mL) but was less toxic (IC50 1100 ng/mL) yielding an improved selectivity index of 180 (D6 clone) and 140 (W2 clone). Nitration at the 6-position of 1 resulted in a slight decrease in the activity (IC50 18 ng/mL, 8) while nitration at the 8-position decreased the activity significantly (IC50 310 ng/mL). Introduction of a 6-methoxyl group (21a) also decreased the activity (IC50 28 ng/mL). The methyl ester group in the 3-position in the β-carboline moiety (21b) retained good activity (IC50 11 ng/mL). Reduction of the pyridine ring in the β-carboline moiety (20a-d) also decreased the activity. The above results indicated that modification of the β-carboline ring is tolerated. Further modification may result in a lead with better therapeutic index.
Reduction of the double bonds at 15 and 32 resulted in differences in activity. When just the double bond at C-32 in the bottom eight-membered ring of 1 or 2 was reduced (5 and 6), the activity was reduced significantly (IC50 200 ng/mL). A second reduction, of the double bond at C-15 gave 3 and 4 and increased activities with IC50 values of 82 and 90 ng/mL, respectively. These results indicate that the double bond at C-32 is important for the activity (either bonding or conformation) while the double bond at 15 is not required. Selectively reducing the double bond at C-15 in 1 and 2 may result in more potent antimalarial products.
Compound 14, differing from 2 by an additional carbonyl functionality at position 31 and lack of a double bond at position 32 in the bottom eight-membered ring, only showed marginal antimalarial activity (IC50 780 ng/mL), a hundred fold less active than 2. Introduction of an α,β-unsaturated system with carbonyl functionality intact (17a-h) did not improve the antimalarial activity, nor did reduction of the carbonyl group (16). Modification of the carbonyl functional group with hydrazone (15) or alkylation (18) resulted in significant improvement in antimalarial activity. Manzamine F-31-hydrazone (15) showed an IC50 of 29 ng/mL against P. falciparum (D6 clone), which is a ca. 27 fold increase in activity over the mother compound. 18 showed an IC50 of 77 ng/mL, an one order of magnitude increase. Neither compound showed toxicity against Vero cells at the highest tested concentration of 4.6 μg/mL. The improved activity of 14 analogues clearly showed that the carbonyl functional group is a liability for the bioactivity and a lipophilic functionality is preferred for the antimalarial activity.
Antibacterial and antifungal activity
In addition to the antiprotozoal assays, in vitro antimicrobial activities of the manzamine analogues against Mycobacterium tuberculosis, Candida albicans, Cryptococcus neoformans, and Mycobacterium intracellulare were investigated. Manzamines showed moderate activity against Mycobacterium tuberculosis (H37Rv), of which 2 exhibited the best activity with an MIC of 0.9 μg/mL (see Table 2). In contrast, manzamine derivatives exhibited highly potent activity against M. intracellulare. Compounds 21b and 20d showed the most potent activity with IC50 values of < 0.02 and 0.06 μg/mL, which are about one order of magnitude more potent than 1 and the positive control ciprofloxacin. 2, tetrahydro-8-hydroxymanzamine A (4), manzamine D (20a), 8-acetoxymanzamine A (9), 6-methoxymanzamine D (20c), 6-nitromanzamine A (8), and 8-nitromanzamine A (7) showed slightly improved activity compared to 1 and ciprofloxacin with IC50 values of 0.1∼0.25 μg/mL, indicating that the β-carboline ring can be modified to yield improved activity against M. intracellulare. The decreased activity of the 9-N alkylation products of 1 (6a-g) revealed that the free NH is required for the activity. Modification of the carbonyl group of 14 gave varied results. While the α,β-unsaturated derivatives of 14 (17a-g) diminished the activity, 15 and 18 exhibited potent activity with IC50 values of 0.09 and 0.25 μg/mL, respectively, more than one order of magnitude more potent than 14.
Table 2.
In Vitro Antibacterial and Antifungal Activities*
| Compounds | M. tuberculosis (H37Rv) | C. albicans | C. neoformans | M. intracellulare |
|---|---|---|---|---|
| MIC (μg/mL) | IC50 (μg/ml) | IC50 (μg/ml) | IC50 (μg/ml) | |
| 1 | 1.5 | 2.0 | 1.5 | 0.35 |
| 2 | 0.9 | 3.5 | 2.0 | 0.1 |
| 3 | 50 | 7.5 | 0.9 | 0.45 |
| 4 | 24 | 6 | 1.5 | 0.1 |
| 20a | 0.99 | 15 | 0.8 | 0.1 |
| 20c | >64 | >20 | 2.0 | 0.2 |
| 20d | >64 | >20 | 3.5 | 0.06 |
| 21b | >64 | 20 | 2.0 | <0.02 |
| 15 | 1.9 | >20 | 1.0 | 0.09 |
| 18 | 1.9 | 2.5 | 6.0 | 0.25 |
| 9 | NT | 1.5 | 1.5 | 0.10 |
| 8 | 1.6 | >20 | 2.5 | 0.15 |
| 7 | NT | >20 | 4.0 | 0.25 |
| 14 | NT | 15 | 6.5 | NT |
| Amphotericin B | NT | 0.20 | 0.75 | NT |
| Ciprofloxacin | NT | NT | NT | 0.30 |
| Rifampin | 0.09 | NT | NT | NT |
For a full list of compounds and activity data, please see supporting material. NT = not tested.
All the analogues were evaluated for activity against Cryptococcus neoformans and improved activities were observed with several of the analogues synthesized for this study. Compounds 3, 20a, and 15 showed potent activity (IC50 0.9, 0.8, 0.9, and 1.0 μg/mL, respectively), comparable with that of amphotericin B (IC50 0.75 μg/mL), and can be considered as leads. The remaining compounds are less activity than 1.
1 showed moderate active against Candida albicans. Modification resulted decreasing of activity for all products.
Anti-neuroinflammatory activity
The in vitro anti-neuroinflammatory activity of natural and synthetic analogues of manzamines was investigated on phorbol 12-myristate 13-acetate-stimulated generation of superoxide anion (O2-) and thromboxane B2 (TXB2) from activated rat neonatal microglia as previously described.34,13 Lactate dehydrogenase (LDH) was used as a marker for cell cytotoxicity.35 As shown in Table 3, 21b potently inhibited O2- (IC50=0.03 μM) and TXB2 (IC50=0.11 μM) release from brain microglia, followed by 6g, 21a and 6d. Furthermore, minimal cytotoxicity, determined as lactate dehydrogenase release, was observed for these compounds or for 1 at the highest concentration tested in vitro (10 μM). This data thus supports the hypothesis that these manzamine compounds may be potential leads for the development of novel agents to modulate activated microglia cells in neuroinflammatory disorders such as Alzheimer's disease and malaria.10,11,36,37,38 All other N-alkylation products (compounds 6a, b, c, d, e, f) exhibited decreased activity on microglia O2- and TXB2 generation. Hydrogenation (3, 4) also appeared to decrease the inhibitory activity on O2- and TXB2 generation. 20a derivatives showed both less activity in the anti-neuroinflammatory assay and increased cytotoxicity to microglia cells.
Table 3.
In Vitro Anti-neuroinflammatory Activity*
| Compounds | O2- | TXB2 | LDH |
|---|---|---|---|
| IC50(μM) | IC50(μM) | LDH50(μ,M) | |
| 1 | 0.1 | <0.1 | >10 |
| 6d | 0.12 | 0.4 | >10 |
| 6f | >10 | 3.2 | >10 |
| 6g | 0.07 | 0.07 | >10 |
| 21a | 0.1 | 0.11 | >10 |
| 21b | 0.03 | 0.11 | >10 |
Anti-neuroinflammatory assay: effect of compounds on rat microglia PMA [1 μM]-stimulated release of O2-, TXB2 and LDH. Data shown corresponds to 2-7 independent experiments and is expressed as IC50 (μM) for O2- and TXB2. LDH50 (μM) is the compound's concentration causing 50% percent of maximal LDH release triggered by treating microglia with 0.1% Triton X-100. For a full list of compounds and activity data, please see supporting material.
Blood-Brain Barrier Permeability
Due to the efficacy of 1 and its analogs in neuroinflammation and against infectious agents that could cause cerebral infections (e.g. Cryptococcus and Plasmodium), the transport of 1 was evaluated across MDR-MDCK monolayer - a model for in vitro BBB transport study. The permeability of the compound was observed in both absorptive and secretory directions but the efflux ratio was < 1 indicating no net efflux. The transport was linear with time (Figure 1 A and B) in both directions but drug permeated at a higher rate from the apical side to the basolateral side (absorptive direction) of the monolayer. The permeability of 1 HCl salt in the absorptive direction (Papp 1.9 × 10-6 cm/sec) was much higher than that of its free base form (Papp 0.87 × 10-6 cm/sec) as shown in Figure 2. However, the permeability of 1 was much lower in comparison to caffeine (Papp 17 × 10-6 cm/sec) which is a highly permeable drug. That 1 can permeate across the BBB strengthens the therapeutic value of this class of compounds, which already have promising anti-infectious and anti-neuroinflammatory activities and significant oral bioavailability.
Figure 1.
Time dependent transport of 1 salt and its free base (50 μM) across MDR-MDCK monolayer
Figure 2.
Permeability Coefficient of 1 salt and its free base in absorptive and secretory directions of MDR-MDCK monolayer.
In vivo Antimalarial Evaluation of 1 and 82
Initial in vivo evaluations of 1 were carried out with the assistance of Tropical Disease Research at the Swiss Tropical Institute in Basel and Northwick Park Institute for Medical Research, Harrow UK, through a WHO contract. 1 has shown ED50/ED90 values of 0.57/1.7 mg/kg P.O. in Plasmodium yoelii infected mice, which were significantly better than sodium artesunate which had ED50/ED90 value of 4.1/17.0 mg/kg. Administration of 3×5 mg/kg of 1 subcutaneously or orally to mice inoculated with P. berghei resulted in prolonged mean survival times of 24.3 and 19.7 days in comparison with 14.3 days for chloroquine at 3×10 mg/kg P.O.
In our in vivo experiments 1 was orally administered to mice from day one after the infection at three doses, 3×1.1 mg/kg, 3×3.3 mg/kg, and 3×10 mg/kg. 1 was able to produce a 75% reduction of the parasitemia level and significantly increase the survival time of mice infected with P. berghei with a dose of as little as 1.1 mg/kg (Table 4). A 75% cure rate of infected mice was achieved by a treatment with 3×10 mg/kg of 1, while 25% cure of the infected animals was seen at 3×3.3 mg/kg. This antimalarial activity of 1 in mice was better than chloroquine at a similar dose.
Table 4.
Multiple Dose Treatments of Manzamine A in Plasmodium berghei Infected Mice
| Compounds (dose) | Inhibition of Parasitemia (%)a | Average % Parasitemia (days post infection) |
MST Days (%)b | Cure (%)c | ||||
|---|---|---|---|---|---|---|---|---|
| 7 | 10 | 14 | 21 | 28 | ||||
| Control | - | 16.7 | 22.1 | - | - | - | 11.8 (0) | 0 |
| 1 (3×1.1 mg/Kg) | 75 | 5.5 | 11.5 | 21.7 | 39.6 | 58.6 | 26.5(50) | 0 |
| 1 (3×3.3 mg/Kg) | 88 | 2.8 | 4.3 | 11.9 | 21.6 | 53.9 | >30 (100) | 25 |
| 1 (3×10 mg/Kg) | 98 | 0.2 | 0.4 | 0.9 | 1.9 | 3.3 | >30(100) | 75 |
| Chloroquine (3×5 mg/Kg) | 88 | 4.5 | 7.8 | 13.5 | 21.7 | 39.3 | >30 (100) | 0 |
% decrease in level of parasitemia in mice on day 5 post infection, as compared to untreated controls.
MST-mean survival time in days (% of mice remaining alive until day 28 post infection)
Mice without parasitemia until 28 days after the infection were considered as cure.
We have shown that 1 has good oral bioavailability, a long half-life of 54 hours and low plasma clearance.14 This facilitates the possible clearance of malaria with a single treatment of 1. For this reason single dose schedules of 1 in mice infected with malaria were evaluated. A single dose of 50 mg/kg showed complete clearance of parasitemia until day 14 post infection and was able to produce a 60% cure rate. Dosing with 100 mg/kg or higher was found to cure all the mice infected with P. berghei (Table 5). In contrast, treatment with quinine (200 mg/kg) failed to cure any mice while chloroquine (200 mg/kg) only produced an 80% cure-rate. The untreated infected mice showed some initial weight gain followed by marked weight loss about 10 days after the inoculation, which correlated with the development of parasitemia. Treatment with 1 caused mild weight loss initially until day 5 followed by normal weight gain thereafter. Mice treated with 100 mg/kg of 1 did not show any behavioral signs of toxicity. Mice treated with higher doses of 200 or 300 mg/kg began to exhibit signs of toxicity beyond weight loss. A successful single dose treatment for malaria would be highly significant because of its potential to reduce the onset of drug-resistance associated with non-compliance of multiple dose schedules.
Table 5.
Oral Single Dose Treatment of manzamine A on Plasmodium berghei Infection in Mice
| Compound (dose) | Activity (%)a | Average Parasitemia (%) Days post infection |
Cure (%)b | |||||
|---|---|---|---|---|---|---|---|---|
| 5 | 7 | 10 | 14 | 21 | 28 | |||
| Control | - | 9.8 | 15.9 | 21.7 | 39.8 | - | - | - |
| 1 (50 mg/kg) | 100 | 0 | 0 | 0.4 | 1.0 | 5.2 | 6.25 | 60 |
| 1 (100 mg/kg) | 100 | 0 | 0 | 0 | 0 | 0 | 0 | 100 |
| 1 (200 mg/kg) | 100 | 0 | 0 | 0 | 0 | 0 | 0 | 100 |
| Chloroquine (200 mg/kg) | 100 | 0 | 0 | 0 | 0.2 | 4.0 | 8.0 | 80 |
| Quinine (200 mg/kg) | 26 | 7.3 | 11.6 | 14 | 32 | 45 | - | 0 |
% Decrease in level of parasitemia in mice on day 5 post infection, as compared to untreated controls.
Percentage of mice without parasitemia until day 28 post infection.
Intraperitoneal administration of 1 and 2 (10×3 mg/kg) in mice infected with malarial parasites was also evaluated (see Table 6). 1 possesses strong antimalarial activity in vivo with 100% suppression of the parasitemia at day 5, but it is toxic at 10 mg/kg. No immediate adverse/toxic reactions were noticed until day 3 post treatment with 1. 2 produced a remarkable 80% cure with mild reversible weight loss. This result is consistent with the in vitro data, which indicated lower toxicity of 2 relative to 1 in Vero cells.
Table 6.
Antimalarial Activity of Manzamine A and 8-Hydroxymanzamine A by i.p.
| Treatments | %parasitemia suppressiona | Survivalb Day of the death (MST)c |
Cured | |
|---|---|---|---|---|
| Day 5 | Day 7 | |||
| Vehicle control | 0/5 12/16/12/16/12 (14) |
0/5 | ||
| 1 10×3 mg/kg | 100 | - | 0/5 5*/6*/5*/6*/6* 5.6* |
0/5 |
| 2 10×3 mg/kg | 100 | 100 | 5/5 (>28) | 4/5 |
% Suppression in parasitemia is calculated by considering the mean parasitemia in control group as 100%.
number of animal survived day 28/total animal in group (the day of the death- post infection).
MST- mean survival time (days).
Number of mice without parasitemia (cured) through day 28 post infection.
Death related to toxicity
GSK-3β Docking
Glycogen synthase kinase 3 (GSK-3) was originally identified as a protein kinase involved in the regulation of glycogen metabolism, and now is known to function in a wide range of cellular processes including in the cell life cycle, apoptosis, and development. Aberrant regulation of GSK-3 has been implicated in a range of human pathologies such as Alzheimer's disease, type-2 diabetes, bipolar disorder, and cancer.39,40,41,42 GSK-3 was originally identified in mammals, and homologues have been found in all eukaryotes. Since several of the manzamines were recently discovered to be inhibitors of Homo sapiens GSK-3β (HsGSK-3β),43,20,44, we performed molecular docking studies44 for the representative compound 2 with HsGSK-3β. Building on the recently published evidence that 2 was an ATP-noncompetitive inhibitor of HsGSK-3β,43,20,45 we explored the ability of 2 to bind in a pocket that is located in the vicinity of the activation pocket formed by three basic residues, Arg96, Arg180 and Lys205. In the first X-ray crystal structures of HsGSK-3β46,47 a charged phosphate, sulfate or sulfonate-containing moiety was found to be bound in that vicinity. This pocket was proposed to be the binding site for thiadiazolidinones by Martinez et al. in their reports on the first ATP-noncompetitive inhibitor.48,49 For this work, we used an X-ray crystal structure of HsGSK-3β without a co-crystallized ligand in the ATP-binding site but which had a sulfate coordinated to Arg96, Arg180 and Lys205, with Val204 and Asn203 making fairly close interactions as well (pdb code: 1gng). 47
The docking results showed that 2 fits well into the ATP-noncompetitive pocket (Figures 3 and 4). The three polar interactions exhibited by 2 are shown in Figure 4. The 8-hydroxyl group of 2 formed significant electrostatic interactions with Arg96 and Ala204. The acceptor oxygen of the 8-hydroxyl group interacted with a donor NH from Arg96 whereas the 8-hydroxyl donor hydrogen interacted with both the backbone carbonyl of Ala204 and with an intermediary water molecule which in turn was hydrogen bonded to the NH of Arg96. The tertiary nitrogen of the β-carboline ring of 2 showed water mediated hydrogen bonding with the NH of Asp90. This may partly explain why 20a, which has a reduced β-carboline ring, is less active than 2. A key hydrophobic interaction between the 8-membered ring of 2 and Phe93 was found. This may partly explain why 5, which has a reduced 8-membered ring, is less active than 2. The β-carboline ring also showed hydrophobic interactions with the backbone of Arg96. Thus the combination of three polar interactions and several nonpolar interactions lead to a favorable docking pose for 2 in the ATP-noncompetitive binding site of HsGSK-3β.
Figure 3.
Binding mode of docked 2 (green C, red O, blue N, cyan H) within the ATP-noncompetitive pocket found in the vicinity of Arg96, Arg180 and Lys205. Key amino acids in the binding pocket for ATP-competitive inhibitors (left side of the enzyme) are labeled. (a) Connolly molecular surface of GSK-3β, with surface red and blue regions indicating the presence of hydrogen bond donors and acceptors, respectively, in the protein. (b) Tube model (colors) of the two active sites of GSK-3β (magenta, yellow and cyan indicate α-helices, β-sheets and loops, respectively; protein side-chain gray C, red O, blue N, cyan H).
Figure 4.
Key interactions of 2 within the ATP-noncompetitive pocket of GSK-3β (gray C, red O, blue N, cyan H).
To date, the mechanism for the diverse bioactivities of manzamine alkaloids is still unclear. The above docking results and our recent discovery of 1 as a selective inhibitor of GSK-3β and CDK-5 suggest that GSK-3β and closely related kinases may represent common targets for the diversity of biological activity observed including the antimalarial activity. Recently, Plasmodium falciparum parasite GSK-3 (PfGSK-3) was identified by Droucheau et al. and was suggested to be a possible antimalarial target. The whole protein sequence of PfGSK-3 is 43.1% identical and 58.9% similar to HsGSK-3β (central catalytic domain is 58.2% identical and 76.0% similar).50 Hence it is possible that the antimalarial activity exhibited by manzamine and its analogs is due to inhibition of Pf-kinases. However, further experimental studies are required to prove that the antimalarial activity of manzamines is through the inhibition of Pf-kinases. Although PfGSK-3 shares similarity with human GSK-3, divergent sensitivity to a series of GSK-3β inhibitors was observed, suggesting that selective inhibitors to either PfGSK-3 or HsGSK-3β might be identified.50 The high activity of 1 against P. falciparum parasites and the moderate inhibitory activity of HsGSK-3β by 1 may indicate that 1 is a highly selective inhibitor of PfGSK-3. A new function has been found recently for GSK-3 as a vital factor in the inflammation process.51,52 Thus, the anti-neuroinflammatory activity of manzamines may be also caused by inhibition of GSK-3β. There is no published information about GSK-3 as a target for antibacterial agents but broad-spectrum kinase inhibitors like staurosporine do indeed show antibiotic activity.53,54.
Conclusion
This structure-activity relationship study has allowed us to refine the structural requirements for the unique manzamine alkaloids to act against neuroinflammation and infectious disease including malaria. A number of derivatives exhibited highly promising activity and can be considered as potential drug leads.
Both in vitro and in vivo data clearly revealed significant potential for 1 and 2 analogs in the treatment of malaria. Although toxicity has been seen in high doses and daily dosing schedules of 1, the successful development of commonly utilized antimalarial agents from quinine and quinolines, despite their significant toxicity at higher doses, bodes well for the development of a novel class of antimalarial drugs from manzamines by optimization of both structure and drug administration. This was evidenced by the significantly reduced toxicity of 2 compared to 1 in intraperitoneal administration to mice (Table 6). Highly encouraging results have been found by modification of 14. Two derivatives with modification of the carbonyl group of 14 (15 and 18) showed 10- to 27-fold increase in antimalarial activity without showing toxicity to Vero cells. This suggests that further modification along this direction may result in an optimized candidate with suitably potent antimalarial activity and a better therapeutic window.
One of the most deadly complications of malaria is cerebral malaria, which is fatal in about 30-50% of the cases.36 Although the immunopathogenesis of cerebral malaria remains incompletely understood,55 it appears that one of the earliest events is an increase in the permeability of the blood-brain barrier probably mediated by CD8+ T cell damage to the microvascular endothelium, an event that is followed by leakage of cytokines, malaria antigens and other potentially harmful molecules into the cerebral parenchyma, with concomitant activation of microglia cells.36 Thus a significant pathophysiological alteration observed in human cerebral malaria is widespread activation and proliferation of microglial cells.56,57 The anti-neuroinflammatory and immunosuppressive activity of manzamines13,58 along with their BBB permeation is exceptional for the development of novel treatments of cerebral malaria since currently there is no specific treatment for cerebral malaria.59
Manzamine analogues exhibited selective activity against Mycobacterium intracellulare. Eleven derivatives (2, 4, 7, 8, 9, 15, 18, 20a, 20c, 20d, and 21b) exhibited improved activity compared to manzamine A and the positive control ciprofloxacin. The two derivatives with a 3-methyl carboxylate, 21b and 20d, were 5- to 15-fold more potent than ciprofloxacin. Compounds 20a, 3, and 15 showed potent activity against Cryptococcus neoformans, which are comparable with amphotericin B. These manzamine analogues clearly have great potential as antibiotics or antifungals which are selectively active against M. intracellulare and C. neoformans.
With regard to anti-neuroinflammatory activity, the most effective manzamine derivatives were 21b and 6g, with 21b the best for O2- and 6g best for TXB2 release from brain microglia. 21a and 6d were also highly active. The four compounds showed no toxicity at 10 μM, suggesting they are potentially good leads for the development of novel agents for the control of neuroinflammatory diseases.
Evidence shows that GSK-3 plays a key role in the neurodegenerative diseases including Alzheimer's (AD), Parkinson's and related neurodegenerative diseases and GSK-3 has been considered as one of the most important potential targets for neurodegenerative disease.35,41,42 It is also well established that inflammation occurs in AD and contributes to the pathological progression of AD.60,61,62 Leads like manzamines which have both anti-neuroinflammatory and GSK-3β inhibitory activity would have increased benefits for the treatment of neurodegenerative diseases. Moreover, since GSK-3 is involved in so many pathways for cell function, selective and moderate inhibition is the best way to reach the therapeutic goal and avoid or decrease potential side effects. Manzamines exhibited selective, moderate inhibition to GSK-3β, which for 1 was shown to be in a specific ATP-noncompetitive manner,34 suggesting that they are appropriate candidates for GSK-3β inhibition.
Molecular docking of 2 to HsGSK-3β was used to find a binding pose in the previously identified ATP-noncompetitive binding site, which was stabilized by a combination of three polar interactions and several nonpolar interactions. This opens up the exciting possibility of rational structure-based optimization of manzamines as GSK-3β inhibitors.
Despite the diverse bioactivities which have been shown for manzamine alkaloids, our results clearly show that promising drug leads for treatment of malarial, bacterial, neuroinflammatory and neurodegenerative diseases are achievable by modification and optimization of derivatives of 1 which also shows moderate BBB permeation in an in vitro model. 1 has been shown to be highly stable at room temperature in aqueous solution. No significant decomposition of the compound was observed for over twenty-four months during exposure to light at room temperature in a 0.05 N HCl aqueous solution (20 mg/mL). A putative Micromonospora (Actinomycetes sp.) isolated from the manzamine producing sponge has been patented for manzamine production but validation through incorporation experiments remains a challenge.63 Although considerable effort remains to optimize the culture condition to reach a high yield of manzamines, access to a reliable microbial source for the production of the manzamines may provide potential cost-effective production of manzamines without disruption to fragile marine ecosystem by excessive sponge harvest. As a result there is significant potential for analogs of the manzamines class as antiparasitic-antibiotics and in the control of neuroinflammation. This unique combination of activities could be exploited to control both the pathogen as well as the associated inflammation.
Experimental Section
General Experimental Procedures
The NMR data were recorded in CDCl3 on a Bruker DRX NMR spectrometer operating at 400 MHz for 1H and 75 MHz for 13C. Chemical shift (δ) values are expressed in parts per million (ppm) and are referenced to the residual solvent signals of CDCl3. The HRMS spectra were measured using a Bruker MicroTOF with electrospray ionization. TLC analysis was carried out on silica gel G254 or aluminum oxide ALOX-100 UV254 μm. The purity of each compound were tested to be ≥ 95% by LC/MS using an Agilent HPLC connected to a Bruker MicroTOF with electrospray ionization (Phenomenex Luna C18 4.6×150 mm 5μm column, eluted with gradient acetonitrile:H2O:0.1% formic acid, detect at 235 and 282 nm).
General Preparation of Compounds 6a-i
1 (54.8 mg, 0.1 mmol) was dissolved in 5 mL of dry N,N-dimethylformamide and stirred for 15 min in a Dewar flask at 0°C. To this 95% NaH (2.33mg, 0.12mmol) was added with care and stirred for an additional 5 minutes; methyl iodide (MeI, 15 μL, 0.24 mmol) was added to the reaction mixture at 0°C. After 1 h, the mixture was allowed to warm to room temperature, and was stirred continuously for 2-3 h. The completion of the reaction was monitored by TLC and the reaction mixture was slowly added to a mixture of ice-cold water and stirred for 15 min.
The aqueous layer was extracted with Et2O (2 × 50 mL) and the combined organic layers were washed with brine and dried over anhydrous Na2SO4, with the solvent being removed under reduced pressure. The crude product was chromatographed on a 250 × 10 mm, 5m Luna C8 Phenomenex HPLC column using a water and acetonitrile gradient with the flow rate of 8 mL/min. The remainder of the manzamine A analogues (6b-i) were synthesized using the same reaction conditions.
9N-Methylmanzamine A (6a): yield 84 %; 1H NMR (CDCl3) δ 8.40 (d, J = 5.2), 7.78 (d, J = 5.2), 7.51 (d, J = 7.8), 7.36 (t, J = 8.0), 7.56 (d, J = 7.4), 7.59 (d, J = 8.0), 5.78 (s), 1.59 (m), 2.23 (m), 2.42 (m), 2.79 (m), 5.54 (brs), 5.30 (brs), 1.55 (m) 1.81(m), 1.30 (m), 1.56 (m), 1.42 (m), 1.50 (m), 2.31 (m), 2.82 (m), 2.07 (m), 2.94 (m), 2.45 (m), 2.71 (m), 2.29 (m) 3.76 (s), 2.79 (m), 3.19 (m), 1.70 (m), 1.79 (m), 1.23 (m), 1.70 (m), 1.58 (m), 1.79 (m), 5.90 (brs), 5.66 (brs), 3.05 (m), 2.30 (m), 2.43 (m), 2.64 (d, J = 11.3), 2.21 (d, J = 11.3), 3.87 (s); 13C NMR (CDCl3) δ 142.9, 142.8, 139.5, 138.1, 136.5, 134.8, 130.5, 130.0, 129.2, 128.4, 128.1, 127.4, 123.2, 120.7, 120.5, 113.8, 113.4, 75.0, 70.1, 69.5, 63.8, 58.9, 53.6, 52.3, 51.8, 44.1, 37.9, 37.6, 33.6, 32.7, 31.7, 29.6, 28.0, 27.1, 26.7, 24.6, 20.3; HRESIMS m/z calcd for C37H47N4O [M+H]+ 563.3750, found 563.3764.
9N-Ethylmanzamine A (6b): yield 87 %; 1H NMR (CDCl3) δ 8.38 (d, J = 5.2) 7.72 (d, J = 5.2), 7.57 (d, J = 7.8), 7.38 (t, J = 8.0) 7.51 (d, J = 7.4), 7.59 (d, J = 8.0), 5.73 (s), 1.56 (m), 2.24 (m), 2.47 (m), 2.76 (m) 5.55 (brs), 5.23 (brs), 1.52 (m), 1.78 (m), 1.54 (m), 1.67 (m), 1.76 (m), 1.56 (m), 2.67 (m), 2.78 (m), 2.02 (m), 2.95 (m), 2.44 (m), 2.77 (m), 2.26 (m), 3.75 (s),2.74 (m), 3.23 (m), 1.67 (m), 1.76 (m), 1.26 (m), 1.75 (m), 1.54 (m), 1.75 (m), 5.94 (brs), 5.67 (brs), 3.04 (m), 2.36 (m), 2.46 (m), 2.63 (d, J = 11.3), 2.27 (d, J = 11.3), 3.84 (m), 1.53 (m); 13C NMR (CDCl3) δ 142.7, 142.6, 139.7, 138.4, 136.5, 134.6, 130.3, 130.1, 129.1, 128.2, 128.2, 127.5, 123.3, 120.7, 120.4, 113.6, 113.2, 75.2, 70.4, 69.4, 63.7, 58.9, 53.5, 52.4, 51.7, 47.8, 44.3, 37.6, 37.5, 32.5, 31.5, 29.7, 28.1, 27.3, 26.4, 24.5, 20.2, 14.1; HRESIMS m/z calcd for C38H49N4O [M+H]+ 577.3906, found 577.3911.
9N-Propylmanzamine A (6c): yield 82 %; 1H NMR (CDCl3) δ 8.42 (d, J = 5.2), 7.81 (d J = 5.2), 7.69 (d, J = 7.8), 7.34 (t, J = 8.0), 7.53 (d, J = 7.4), 7.57 (d, J = 8.0), 5.83 (s), 1.42 (m), 2.13 (m), 2.30(m), 2.61 (m), 5.59 (brs), 5.34 (brs), 1.57 (m), 1.77 (m), 1.37 (m), 1.62 (m), 1.48 (m), 1.51 (m), 2.38 (m), 2.73 (m), 2.01 (m), 2.99 (m), 2.30 (m), 2.62 (m), 2.14 (m), 3.78 (s), 2.68 (m), 3.15 (m), 1.82 (m), 1.95 (m), 1.35 (m), 1.67 (m), 1.51 (m), 1.66 (m), 5.92(brs), 5.63 (brs), 3.11 (m), 2.25 (m), 2.56 (m), 2.62 (d, J = 11.3), 2.30 (d, J = 11.3), 3.83 (m), 1.81 (m), 1.27 (m); 13C NMR (CDCl3) δ 142.7, 142.6, 139.4, 138.3, 136.8, 134.8, 130.3, 130.2, 129.6, 128.4, 128.1, 127.3, 123.1, 120.8, 120.6, 113.7, 113.5, 75.0, 70.6, 69.5, 63.4, 58.6, 54.2, 53.3, 52.7, 51.4, 44.4, 37.5, 37.3, 32.8, 31.4, 29.6, 28.4, 27.5, 26.6, 24.3, 21.3, 20.7, 13.8; HRESIMS m/z calcd for C39H51N4O [M+H]+ 591.4063, found 591.4067.
9N-butylmanzamine A (6d): yield 87 %; 1H NMR (CDCl3) δ 8.48 (d, J = 5.2), 7.78 (d, J = 5.2), 7.51 (d, J = 7.8), 7.34 (t, J = 8.0), 7.58 (d, J = 7.4), 7.59 (d, J = 8.0), 5.72 (s), 1.56 (m), 2.23 (m), 2.47 (m), 2.79 (m), 5.54 (brs), 5.30 (brs), 1.59 (m), 1.81 (m), 1.30 (m), 1.56 (m), 1.43(m), 1.50 (m), 2.31 (m), 2.86 (m), 2.08 (m), 2.94 (m), 2.45 (m), 2.71 (m), 2.25 (m), 3.77(s), 2.77 (m), 3.19 (m), 1.70(m), 1.79 (m), 1.29 (m), 1.70(m), 1.54 (m), 1.75 (m), 5.90 (brs), 5.66 (brs), 3.09 (m), 2.30 (m), 2.43 (m), 2.64 (d, J = 11.3), 2.21 (d, J = 11.3), 3.87 (m), 1.79 (m), 1.42 (m), 1.18 (m); 13C NMR (CDCl3) δ 142.7, 142.5, 139.3, 138.2, 136.7, 134.5, 130.1, 130.1, 129.3, 128.4, 128.2, 127.5, 123.4, 120.6, 120.4, 113.5, 113.4, 75.2, 70.3, 69.6, 63.8, 58.7, 57.1, 53.6, 52.4, 51.2, 44.6, 37.7, 37.5, 32.5, 31.3, 29.8, 29.8, 28.2, 27.6, 26.7, 24.6, 20.3, 19.1, 13.3; HRESIMS m/z calcd for C40H53N4O [M+H]+ 605.4219, found 605.4222.
9N-isobutylmanzamine A (6e): yield 82 %; 1H NMR (CDCl3) δ 8.47 (d, J = 5.2), 7.74 (d, J = 5.2), 7.57 (d, J = 7.8), 7.35 (t, J = 8.0), 7.57 (d, J = 7.4), 7.54 (d, J = 8.0), 5.75 (s), 1.56 (m), 2.24 (m), 2.46 (m), 2.74 (m), 5.55 (brs), 5.30 (brs), 1.56 (m), 1.85 (m), 1.33 (m) 1.55 (m), 1.47 (m), 1.54 (m), 2.36(m), 2.84 (m), 2.07 (m), 2.93 (m), 2.46 (m), 2.74 (m), 2.26 (m), 3.75 (s), 2.74 (m), 3.16 (m), 1.73 (m), 1.73 (m), 1.26 (m), 1.74 (m), 1.58 (m), 1.74 (m), 5.94 (brs), 5.64 (brs), 3.06 (m), 2.34 (m), 2.43 (m), 2.64 (d, J = 11.3), 2.23 (d, J = 11.3), 3.82 (m), 1.76 (m), 1.23 (m), 1.23(m); 13C NMR (CDCl3) δ 142.4, 142.2, 139.2, 138.1, 136.8, 134.7, 130.5, 130.4, 129.1, 128.4, 128.2, 127.7, 123.2, 120.6, 120.6, 113.6, 113.3, 75.8, 70.5, 69.3, 63.6, 61.5, 58.4, 53.5, 52.2, 51.8, 44.2, 37.3, 37.2, 32.6, 31.6, 29.5, 28.3, 27.4. 27.2, 26.8, 24.8, 20.1, 18.6 (2C); HRESIMS m/z calcd for C40H53N4O [M+H]+ 605.4219, found 605.4225.
9N-isopentylmanzamine A (6f): yield 84 %; 1H NMR (CDCl3) δ 8.40 (d, J = 5.2), 7.79 (d, J = 5.2), 7.51 (d, J = 7.8), 7.30 (t, J = 8.0), 7.56 (d, J = 7.4), 7.59 (d, J = 8.0), 5.78 (s), 1.53 (m), 2.21 (m), 2.47 (m) 2.79 (m), 5.54 (brs), 5.33 (brs), 1.55 (m), 1.85 (m), 1.33 (m), 1.59 (m), 1.44 (m), 1.53 (m), 2.36 (m), 2.82 (m), 2.07 (m), 2.94 (m), 2.46 (m), 2.75 (m), 2.28 (m), 3.72 (m), 2.75 (m), 3.16 (m), 1.78 (m), 1.77 (m), 1.23 (m), 1.70 (m), 1.58 (m), 1.79 (m), 5.92 (brs), 5.64 (brs), 3.05 (m), 2.33 (m), 2.43 (m), 2.62 (d, J = 11.3), 2.23 (d, J = 11.3), 3.85 (m), 1.72 (m), 1.85 (m), 1.09 (m), 1.09 (m); 13C NMR (CDCl3) δ 142.8, 142.2, 139.6, 138.0, 136.7, 134.5, 130.3, 130.2, 129.7, 128.3, 128.2, 127.3, 123.6, 120.5, 120.4, 113.6, 113.6, 75.5, 70.6, 69.7, 63.8, 59.3, 58.2, 53.8, 52.1, 51.6, 44.1, 37.6, 37.3, 33.5, 32.4, 31.8, 29.7, 28.6, 27.4, 26.7, 26.6, 24.6, 21.8, 21.8, 20.4; HRESIMS m/z calcd for C41H55N4O [M+H]+ 619.4376, found 619.4381.
9N-neopentylmanzamine A (6g): yield 85 %;1H NMR (CDCl3) δ 8.45 (d, J = 5.2), 7.82 (d, J = 5.2), 7.66 (d, J = 7.8), 7.34 (t, J = 8.0), 7.52 (d, J = 7.4), 7.56 (d, J = 8.0), 5.83 (s), 1.42 (m), 2.17 (m), 2.32 (m), 2.61 (m), 5.58 (brs), 5.34 (brs), 1.56 (m), 1.77 (m), 1.38 (m), 1.63 (m), 1.45 (m), 1.51 (m), 2.37 (m), 2.73 (m), 2.04 (m), 2.97 (m), 2.30 (m), 2.63 (m), 2.14 (m), 3.76 (s), 2.65 (m), 3.13 (m), 1.82 (m), 1.97 (m), 1.32 (m), 1.67 (m), 1.53 (m), 1.66 (m), 5.92 (brs), 5.63 (brs), 3.14 (m), 2.25 (m), 2.55 (m), 2.61 (d, J = 11.3), 2.33 (d, J = 11.3), 3.81 (m), 1.05 (s), 1.05 (s) 1.05 (s); 13C NMR (CDCl3) δ 142.9, 142.2, 139.9, 138.5, 136.4, 134.4, 130.7, 130.6, 129.3, 128.9, 128.3, 127.2, 123.4, 120.9, 120.7, 113.9, 113.3, 75.1, 70.2, 69.5, 68.3, 63.6, 58.5, 53.5, 52.7, 51.9, 44.6, 37.9, 37.0, 32.7, 31.4, 29.1, 28.9, 27.6, 27.2, 27.1(3C), 26.8, 24.3, 20.6; RESIMS m/z calcd for C41H55N4O [M+H]+ 619.4376, found 618.4382.
9N-dodecylmanzamine A (6h): yield 81%; 1H NMR (CDCl3) δ 8.40 (d, J = 5.2), 7.78 (d, J = 5.2), 7.51 (d, J = 7.8), 7.36 (t, J = 8.0), 7.56 (d, J = 7.4), 7.59 (d, J = 8.0), 5.78 (s), 1.59 (m), 2.23 (m), 2.42 (m), 2.79 (m), 5.54 (br s), 5.30 (br s), 1.55 (m), 1.81 (m), 1.30 (m), 1.56 (m), 1.42 (m), 1.50 (m), 2.31 (m), 2.82 (m), 2.07 (m), 2.94 (m), 2.45 (m), 2.71 (m), 2.29 (m), 3.76 (s), 2.79 (m), 3.19 (m), 1.70 (m), 1.79 (m), 1.23 (m), 1.70 (m), 1.58 (m), 1.79 (m), 5.90 (brs), 5.66, (brs), 3.05 (m), 2.30 (m), 2.43 (m), 2.64 (d, J = 11.3), 2.21 (d, J = 11.3), 3.87 (s); 13C NMR (CDCl3) δ 142.7, 142.6, 139.2, 138.3, 136.8, 134.7, 130.5, 130.4, 129.8, 128.7, 128.5, 127.5, 123.5, 120.9, 120.8, 113.8, 113.7, 75.4, 70.5, 69.4, 63.5, 58.8, 57.8, 53.5, 52.2, 51.6, 44.4, 37.5, 37.4, 32.9, 31.8, 31.4 (4), 31.1, 30.3, 29.8, 29.3, 28.4, 27.8, 26.5, 26.3, 24.6, 24.4, 22.8, 20.8, 13.8; HRESIMS m/z calcd for C48H69N4O [M+H]+ 717.5471, found 717.5478.
9N-(4-methylcarboxybutyl)manzamine A (6i): yield 76 %; 1H NMR (CDCl3) δ 8.42 (d, J = 5.2), 7.81 (d, J = 5.2), 7.69 (d, J = 7.8), 7.34 (t, J = 8.0), 7.53 (d, J = 7.4) 7.57 (d, J = 8.0), 5.83 (s), 1.42 (m), 2.13 (m), 2.30 (m), 2.61 (m), 5.59 (brs), 5.34 (brs), 1.57 (m), 1.77 (m), 1.37 (m), 1.62 (m), 1.48 (m), 1.51 (m), 2.38 (m), 2.73 (m), 2.01 (m), 2.99 (m), 2.30 (d, J = 11.3), 3.85 (t, J = 6.1), 1.68 (m), 1.57 (m), 2.31 (m), 3.92 (s); 13C NMR (CDCl3) δ 174.8, 144.8, 144.2, 142.3, 138.6, 137.3, 134.1, 132.7, 129.9, 129.6, 128.9, 128.3(2), 122.0, 121.8, 120.0, 113.6, 112.0, 75.2, 69.2, 68.7, 63.6, 53.1, 52.7, 49.9, 49.3, 48.2, 46.0, 44.8, 42.0, 41.2, 35.8, 33.8, 29.7, 28.5, 26.9, 25.9, 25.8, 25.6, 25.4, 21.3; HRESIMS m/z calcd for C42H55N4O3 [M+H]+ 663.4274, found 663.4291.
General Preparation of Compounds 3, 4, 5 and 12
1 (54.8 mg, 0.1 mmol) was dissolved in 5 mL of ethanol, to which was added 5 mg of Palladium charcoal, and stirred for 4 h under H2 gas. The completion of the reaction was monitored by TLC and the solvent was removed in vacuo. The crude reaction mixture was chromatographed on a silica gel column using hexane-ethyl acetate gradient yielding 3 as a major compound (68.3%) and 5 as a minor compound (6.5%). The similar reaction procedure has been applied to 8-hydroxymanzamine A yielding 4 as a major compound (51.7 %) and 12 as a minor compound (1.6 %).
Tetrahydromanzamine A (3): yield 68.3%; 1H NMR (CDCl3) δ 8.64 (d, J = 5.0), 7.83 (d, J = 5.1), 8.09 (d, J = 7.7), 7.26 (dd, J = 7.6, 7.5), 7.53 (dd, J = 7.6, 7.5), 7.58 (d J = 7.5), 6.61 (s), 2.02 (m), 1.83 (m), 2.30 (m), 2.21 (m), 1.53 (m), 1.68 (m), 2.57 (m), 1.57, (m), 1.70 (m), 1.72 (m), 2.68 (m), 2.43 (m), 2.80 (m), 1.99 (m), 2.30 (m), 3.14 (m), 3.78 (s) 3.38 (m), 2.95 (m), 1.80 (m), 1.65 (m), 1.55 (m), 1.77 (m), 2.15 (m), 1.55 (m), 1.65 (m), 3.11 (m), 1.85 (m), 1.56 (m), 2.62 (brd, J = 11.9), 2.30 (d, J = 12.0); 13C NMR (CDCl3) δ 142.3, 142.1, 139.4, 138.6, 136.9, 134.7, 131.4, 127.6, 123.4, 120.9, 120.7, 113.2, 112.9, 75.6, 70.4, 69.3, 63.3, 58.5, 53.3, 52.7, 51.6, 44.7, 37.6, 37.4, 34.1, 32.5, 32.3, 31.7, 31.7, 29.2, 28.0, 27.8, 27.2, 26.2, 24.9, 20.2; HRESIMS m/z calcd for C36H49N4O [M+H]+ 553.3906, found 553.3922.
Dihydromanzamine A (5): yield 6.5 %; 1H NMR (CDCl3) δ 8.56 (d, J = 5.0), 7.87 (d, J = 5.1), 8.25 (d, J = 7.7), 7.21 (dd, J = 7.6, 7.5), 7.49 (dd, J = 7.6, 7.5), 7.46 (d, J = 7.5), 6.59 (s), 2.10 (m), 1.87 (m), 2.36 (m), 2.22 (m), 1.52 (m), 1.63 (m), 2.52 (m), 1.53 (m), 1.79 (m), 1.79 (m), 2.61 (m), 2.47 (m), 2.87 (m), 1.93 (m), 2.33 (m), 3.18 (m), 3.72 (s), 3.31 (m), 2.97 (m), 1.84 (m), 1.67 (m), 1.53 (m), 1.73 (m), 5.89 (m), 5.61 (m), 3.19 (m) 1.81 (m), 1.53 (m), 2.61 (brd, J = 11.9), 2.36 (d, J = 12.0); 13C NMR (CDCl3) δ 142.9, 142.8, 139.8, 137.8, 136.2, 134.2, 130.6, 130.2, 129.7, 127.8, 123.7, 120.7, 120.2, 113.5, 113.2, 75.2, 70.8, 69.8, 63.8, 58.3, 53.9, 52.1, 51.3, 44.4, 37.8, 37.3, 32.9, 32.6, 31.8, 31.6, 28.6, 28.4, 27.6, 26.2, 24.4, 20.9; HRESIMS m/z calcd for C36H47N4O [M+H]+ 551.3750, found 551.3758.
Tetrahydro-8-hydroxymanzamine A (4): yield 51.7 %; 1H NMR (CDCl3) δ 8.40 (d, J = 5.2), 7.78 (d, J = 5.2), 7.56 (d, J = 7.7), 7.03 (dd, J = 7.7, 7.5), 6.96 (d, J = 7.5), 6.78 (s), 2.07 (m), 1.60 (m), 1.42 (m), 1.73 (m), 1.58 (m), 1.55 (m), 1.64 (m), 1.43 (m) 2.45 (dd, J = 12.0, 5.3), 2.32 (m), 2.97 (m), 2.34 (m), 2.05 (m), 1.74 (m), 3.36 (m), 3.76 (s), 3.44 (m), 2.97 (m), 1.70 (m), 2.05 (m), 1.75 (m), 1.58 (m), 1.75 (m), 1.72 (m), 2.99 (m), 2.40 (m), 2.62 (d, J = 11.2), 2.18 (d, J = 11.2); 13C NMR (CDCl3) δ 142.1, 139.1, 138.2, 136.6, 135.4, 132.7, 130.1, 129.7, 123.5, 120.5, 117.5, 113.5, 110.4, 75.3, 70.2, 69.8, 63.9, 58.2, 53.7, 52.4, 51.6, 44.9, 37.7, 37.7, 33.5, 32.7, 32.1, 31.9, 30.9, 29.3, 28.8, 28.6, 27.9, 26.2, 24.7, 20.3; HRESIMS m/z calcd for C36H49N4O2 [M+H]+ 569.3856, found 569.3961.
Dihydro-8-hydroxymanzamine A (12): yield 16 %; 1H NMR (CDCl3) δ 8.31 (d, J = 5.2), 7.81 (d, J = 5.2), 7.58 (d, J = 7.6), 7.12 (dd, J = 7.7, 7.6), 7.05 (d, J = 7.6), 6.18, (s), 1.55 (m), 1.45 (m), 1.46 (m), 1.55 (m), 1.57 (m), 1.66 (m), 1.70 (m), 2.58 (m), 2.25 (m) 3.11 (m), 2.03 (m), 2.34 (m), 1.67 (m), 1.85 (m), 4.93 (s), 4.65 (brd, J = 4.9), 4.24 (m), 1.70, (m), 2.02 (m), 1.65 (m), 1.50 (m), 5.92 (m), 5.67 (m), 2.99 (m), 2.60 (m), 2.52 (d, J = 11.3) 2.20 (m); 13C NMR (CDCl3) δ 142.6, 142.3, 139.4, 138.7, 136.9, 134.7, 133.9, 130.8, 130.6, 129.6, 127.8, 123.7, 120.3, 120.2, 113.2, 75.2, 70.9, 69.8, 63.5, 58.4, 53.4, 52.7, 51.7, 44.4, 37.9, 37.3, 32.3, 32.2, 31.8, 31.4, 29.4, 28.6, 27.2, 26.9, 24.3, 20.9; HRESIMS m/z calcd for C36H47N4O2 [M+H]+ 567.3699, found 567.3708.
General Preparation of Compounds 10 and 11
1 (54.8 mg, 0.1 mmol) was dissolved in 15 mL of dry acetone, with 16.4 mg (1.2 mmol) of anhydrous K2CO3 and 11 μL of anhydrous acetic anhydride added. The reaction was refluxed for 9 h under nitrogen. The completion of the reaction was monitored by TLC and the solvent was removed in vacuo. Then 100 mL of ethyl acetate was added and washed with equal amount of brine solution. This was repeated twice and organic layer was dried on Na2SO4 (200 g), and the solvent was removed in vacuo. The reaction mixture was chromatographed on a silica gel column using hexane-ethyl acetate gradient yielded compound 10 (49.8 %). The similar reaction procedure has been applied to 8-hydroxymanzamine A (2) to yield the compound 11 (46.4 %).
12,13-Dehydromanzamine A (10): yield 49.8 %; 1H NMR (CDCl3) δ 8.41 (d, J = 5.3), 7.81 (d, J = 5.3), 7.96 (d, J = 7.5), 7.14 (dd, J = 7.6, 7.5), 7.30 (d, J = 7.6), 7.68 (d, J = 7.6), 6.60 (s), 6.45 (dd, J = 8.5, 7.0), 2.12 (m), 5.43 (m), 5.35 (m), 1.89 (m), 1.73 (m) 1.38 (m), 1.92 (m), 2.62 (m), 2.51 (m), 2.71 (m), 1.98 (m), 2.73 (m), 2.41 (m), 3.96 (m), 4.88 (s), 3.85 (dd, J = 14.7, 4.7), 3.34 (dd, J = 14.7, 10.8), 1.38 (m), 1.88 (m), 1.60 (m), 2.15 (m), 5.87 (dd, J = 7.7, 7.6), 5.24 (m), 2.41 (m), 1.46 (d, J = 12.9), 2.62 (d, J = 10.9), 2.51 (d, J = 10.9); 13C NMR (CDCl3) δ 142.9, 142.6, 142.4, 139.7, 138.5, 134.3, 130.8, 130.7, 129.7, 129.3, 127.8, 127.6, 123.6, 120.9, 120.8, 118.3, 115.8, 113.7, 113.7, 75.9, 69.6, 63.4, 58.7, 53.3, 52.9, 51.3, 44.8, 37.3, 32.3, 31.5, 29.3, 28.8, 27.8, 26.5, 24.8, 21.1; HRESIMS m/z calcd for C36H43N4 [M+H]+ 531.3488, found 531.3495.
12,13-Dehydro-8-O-acetylmanzamine A (11): yield 46.4 %; 1H NMR (CDCl3) δ 8.38 (d, J = 5.6), 7.82 (d, J = 5.6), 7.97 (d, J = 7.8), 7.23 (dd, J = 7.8, 7.6), 7.29 (d, J = 7.6), 6.50 (s) 6.43 (dd, J = 8.5, 7.0), 2.21 (m), 1.80 (m), 5.56 (m), 5.50 (m), 1.65 (m), 1.42 (m), 1.30 (m), 1.40 (m), 1.32 (m), 2.60 (m), 2.43 (m), 2.83 (m), 1.95 (m), 2.81 (m), 2.44 (m), 3.50 (s), 3.14 (dd, J = 11.9, 8.7), 2.99 (dd, J = 11.8, 4.7), 1.76 (m), 2.15 (m), 2.05 (m), 2.42 (m), 5.84 (dd, J = 7.7, 7.6), 5.21 (m), 3.99 (dd, J = 8.3, 8.2), 2.42 (m), 1.49 (d, J = 12.9), 2.76 (d, J = 11.5), 2.30 (m), 2.53 (s); 13C NMR (CDCl3) δ 167.8, 142.8, 142.8, 142.6, 139.2, 138.7, 136.9, 134.5, 134.4, 130.6, 130.6, 129.4, 128.3, 128.1, 127.8, 123.9, 120.2, 120.1, 117.4, 113.3, 75.8, 69.5, 63.5, 58.6, 53.9, 52.7, 51.2, 44.7, 37.2, 32.2, 31.4, 29.3, 28.2, 27.8, 26.4, 24.3, 20.2, 17.8; HRESIMS m/z calcd for C38H45N4O2 [M+H]+ 589.3543, found 589.3548.
8-Acetoxymanzamine A (9): see reference. 64
6-Nitro-manzamine A (8) and 8-nitromanzamine A (7): Manzamine A (1) (55 mg) was dissolved in 3 mL of trifluoroacetic acid and cooled to 0°C in ice bath. NaNO2 (11 mg) was added in one portion and stirred at 0°C for 1.5 hour. The mixture was poured into water and the precipitate was collected by centrifuge. The precipitate was separated using silica gel preparative TLC (hexane:acetone:triethylamine 8:2:0.1) to yield 8 (6mg) and 7 (6 mg). 6-Nitro-manzamine A (8): yellow powder; 1H NMR (CDCl3) δ 9.04 (d, J = 2.0), 8.49 (d, J = 5.2), 8.41 (dd, J = 9.2, 2.0), 7.89 (d, J = 5.2), 7.77 (d, J = 9.2), 6.50 (s), 6.19 (s), 5.58 (m), 5.38 (t, 10.8), 4.70 (br), 3.69 (s), 3.25 (t, 11.0), 2.93 (d, J = 9.0), 2.80∼2.20 (m), 2.10∼1.20 (m); 13C NMR (CDCl3) δ 144.5, 144.2, 142.7, 141.2, 141.1, 139.4, 135.7, 134.5, 133.2, 129.7, 127.0, 123.7, 123.5, 121.0, 118.4, 114.2, 112.9, 77.9, 71.2, 70.5, 57.6, 53.7 (2C), 49.4, 47.1, 44.9, 40.8, 39.5, 33.9, 28.7, 26.6, 26.6, 25.2, 24.8, 24.3, 20.9; HRESIMS m/z calcd for C36H44N5O3. [M+H]+ 594.3444, found 594.3435. 8-Nitromanzamine A (7): yellow powder; 1H NMR (CDCl3) δ 10.4 (s), 8.57 (d, J = 5.2), 8.48 (d, J = 8.0), 8.45 (d, J = 8.0), 7.87 (d, J = 5.2), 7.40 (t, 8.0), 6.45 (s), 5.96 (m), 5.69 (m), 5.55(m), 5.32 (t, J = 10), 4.27 (br), 3.58 (s), 3.11 (m), 2.61 (m), 2.50∼1.6 (m), 1.4 (m); 13C NMR (CDCl3) δ 144.6, 1443.6, 142.7, 141.4, 141.2, 139.4, 135.3, 134.6, 133.2, 130.0, 127.1, 123.8, 123.7, 121.1, 118.5, 114.4, 113.0, 77.8, 71.2, 70.6, 57.7, 53.7, 53.72, 53.68, 49.5, 47.1, 44.8, 40.5, 39.8, 34.1, 28.8, 26.7, 26.6, 25.3, 24.9, 24.3, 21.0; HRESIMS m/z calcd for C36H44N5O3. [M+H]+ 594.3444, found 594.3426.
21, 27-N-oxamanzamine A (13): manzamine A (1) (56 mg) and 50 mg of 3-chloroperoxybenzoic acid were dissolved in 1 mL of CH2Cl2. After 20 mg of sodium bicarbonate was added, the solution was stirred overnight at room temperature. The reaction mixture was washed with 5% sodium bicarbonate solution and extracted with chloroform. The chloroform layer was evaporated and the residue was purified using silica gel preparative TLC (CHCl3:MeOH 85:15) to get 13 (20 mg). 21, 27-N-oximanzamine A (13): yellow powder; 1H NMR (CDCl3-CD3OD) δ 9.21 (s), 8.35 (d, J = 5.2), 8.08 (d, J = 7.6), 7.82 (d, J = 5.2), 7.49 (m), 7.26 (t, J = 7.6), 6.21 (dt, J = 10, 7.6), 6.12 (s), 5.71 (t, J = 10), 5.60 (br), 5.44 (br), 4.49 (m), 3.91 (d, J = 13.6), 3.84 (s), 3.35 (m), 3.11 (d, J = 10.4), 2.97 (s), 2.72 (m), 2.51 (m), 2.29 (m), 2.10∼1.39 (m); 13C NMR (CDCl3) δ 142.2, 140.7, 139.7, 137.6, 136.7, 133.4, 133.3, 133.2, 132.3, 130.7, 129.7, 128.8, 126.4, 121.5, 121.1, 120.1, 114.3, 112.0, 74.5, 73.7, 70.4, 61.9, 61.4, 49.6, 46.5, 43.1, 38.8, 37.5, 27.7, 25.7, 23.1, 23.1, 22.2, 19.4; HRESIMS m/z calcd for C36H45N4O3 581.3492 [M+H]+ found 581.3474 [M+H]+, 603.3301 [M+Na]+, 563.3359 [M-H2O]+, 291.1766 [M+2H]2+, 282.1719 [M+2H-H2O]2+.
General Preparation of Compounds 20a-d & 21a-d
Method A: 19, tryptamine derivatives, and molecular sieves were stirred at room temperature in CH2Cl2 or EtOH (3 mL) for 2 h. TFA (50 μL) was then added to the reaction mixture and further stirred for another 3 h at 50°C. The reaction mixture was chromatographed over silica gel immediately to give the Pictet-Spengler product.
Method B: to 19 and tryptamine derivatives in 1 ml anhydrous EtOH were added 0.2 ml of trifluoroacetic acid (TFA). The mixture was then absorbed onto 1.5 g of silica gel. The silica gel mixture was placed in a glass vessel and was irradiated in a microwave oven at 390 W for 10 min. The crude mixture was then eluted from the silica gel using acetone, concentrated, and chromatographed over silica gel to furnish the Pictet-Spengler product.
Manzamine D (20a): 19 (46.3 mg) and tryptamine (31 mg) in CH2Cl2 were reacted as described in method A to give manzamine D (20a, 52.7mg, 86%). Manzamine D (20a): white amorphous; 1H NMR (CDCl3) δ 7.78 (br s), 7.51 (d, J = 7.5), 7.35 (brd, J = 7.5), 7.17 (t, J = 7.5), 7.12(t, J = 7.5), 5.95 (dd, J = 18.7), 5.77 (brs), 5.67 (q, J = 9), 5.55 (td, J = 10, 5), 5.30 (t, J = 10), 4.60 (s), 4.17 (t, J = 7), 3.42 (s), 3.35 (m), 3.04 (m), 2.83(m), 2.75(m), 2.51 (m), and 1.20-2.40 (complex); 13C NMR (CDCl3) δ 141.2, 136.8, 135.6, 134.5, 133.9, 132.2, 129.9, 128.8, 127.8, 121.4, 119.2, 118.0, 111.0, 109.4, 75.3, 69.6, 68.5, 59.7, 54.9, 53.4, 50.8, 49.7, 47.3, 44.6, 43.2, 40.7, 37.8, 33.0, 31.8, 28.2, 26.9, 26.0, 25.7, 22.4, 21.7; HRESIMS m/z calcd for C36H49N4O [M+H]+ 553.3906, found 553.3898.
2N-Methylmanzamine D (20b): 19 (50.2 mg) and (2N)-methyltryptamine (30.2 mg) in CH2Cl2 were reacted as described in method A to give N(2)-methylmanzamine D (20b, 22.5 mg, 33%) and unreacted ircinal A (19, 27.9 mg). N(2)-methylmanzamine D (20b): yellowish oil; 1H NMR (CDCl3) δ 7.47 (br s), 7.46 (d, J = 7.6), 7.31 (brd, J = 7.6), 7.12 (t, J = 7.2), 7.07 (t, J = 7.2), 5.88 (q, J = 8), 5.80 (s), 5.61 (q, J = 9), 5.48 (td, J = 10.5), 5.23 (t, J = 9), 4.16 (brs), 3.82 (s), 3.40 (s), 3.11 (m), 2.99 (m), 2.88 (m), 2.40 (s), and 1.2-2.7 (complex); 13C NMR (CDCl3) δ 141.2, 136.5, 135.9, 134.8, 134.1, 132.3, 129.8, 128.9, 121.5, 119.5, 118.2, 111.3, 108.6, 75.5, 69.5, 68.5, 68.1, 55.3, 53.9, 53.6, 51.2, 49.9, 47.2, 44.6, 41.7, 37.1, 32.4, 32.2, 30.0, 29.5, 28.3, 27.1, 26.3, 25.9, 21.9, 21.6; HRESIMS m/z calcd for C37H51N4O [M+H]+ 567.4063, found 567.4042.
6-Methoxymanzamine D (20c): 19 (38.8 mg) and 5-methoxytryptamine (6, 56.1mg) in CH2Cl2 were reacted as described in method A to give 6-methoxymanzamine D (20c, 48.6mg, 89%). Ircinal A (19, 29.5mg) and 5-methoxytryptamine (26.4mg) in anhydrous EtOH were reacted as described in method B to give 6-methoxymanzamine D (20c, 35.1mg, 83%). 6-Methoxymanzamine D (20c): white amorphous; 1H NMR (CDCl3) δ 7.52 (brs), 7.21 (d, J = 8.8), 6.93 (d, J = 1.6), 6.79 (dd, J = 8.8, 1.6), 5.90 (q, J = 8.3), 5.73 (brs), 5.62 (q, J = 8.5), 5.50 (td, J = 10.4, 4.4), 5.22 (t, J = 9.2), 4.14 (brs), 3.85 (s), 3.33 (m), and 1.00-3.20 (complex); 13C NMR (CDCl3) δ 153.4, 141.1, 136.8, 134.8, 134.5, 132.2, 130.6, 129.9, 128.7, 128.2, 111.6, 111.1, 109.2, 100.4, 75.3, 69.5, 68.5, 59.8, 55.9, 54.8, 53.4, 50.7, 49.6, 47.3, 44.5, 43.2, 40.6, 37.7, 33.0, 31.8, 28.5, 26.8, 25.9, 25.7, 22.5, 21.7; HRESIMS m/z calcd for C37H51N4O2 [M+H]+ 583.4012, found 583.4033.
Methyl manzamine D-3-carboxylate (20d): 19 (83.2mg) and D-tryptophan methyl ester HCl (67.1mg) in EtOH were reacted as described in method A to give 20d (87.6mg, 70%). Ircinal A (19, 105.6 mg) and D-tryptophan methyl ester HCl (67.7mg) in anhydrous EtOH were reacted as described in method B to give 20d (95.4mg, 61%). 20d: white amorphous; 1H NMR (CDCl3) δ 7.60 (brs), 7.48 (d, J = 7), 7.35 (d, J = 7), 7.16 (td, J = 7.1), 7.10 (t, J = 7), 5.90 (q, J = 10), 5.86 (brs), 5.64 (q, J = 8), 5.52 (td, J = 10.4), 5.22 (t, J = 9), 4.75 (brs), 4.17 (t, J = 6), 3.82 (s), 3.37 (br s), 3.12 (brd, J = 15), 3.02 (m), 2.88 (brt, J = 12), 2.65 (d, J = 11), and 1.20-2.55 (complex); 13C NMR (CDCl3) δ 173.4, 171.1, 142.8, 136.2, 132.7, 132.6, 127.5, 127.1, 121.7, 119.3, 117.8, 111.4, 108.7, 70.5, 70.1, 60.4, 59.7, 56.8, 56.2, 53.4, 52.5, 52.1, 49.3, 47.1, 44.2, 40.0, 37.7, 32.9, 28.3, 26.5, 25.7, 25.1, 24.8, 24.6, 21.0, 21.0, 14.2; HRESIMS m/z calcd for C38H51N4O3 [M+H]+ 611.3961, found 611.3947.
General Preparation of Compounds (21a) and (21b)
To a solution of manzamine D derivatives in CH2Cl2 (2 mL) at room temperature was added DDQ (2,3-dichloro-5,6-dicyanobenzo-quinone) in benzene (2 mL) and the mixture was stirred for 1 h. The mixture was then washed with saturated NaHCO3 (5 mL), extracted with CH2C12 (2 × 5mL), dried (MgSO4), and evaporated to give brownish oil. Chromatography of the reaction products over silica gel gave the products.
6-Methoxymanzamine A (21a): 20c (141.52 mg) and DDQ (69.5 mg) were reacted as described above to give 6-methoxymanzamine A (21a, 46.4 mg, 33%). 6-Methoxymanzamine A (21a): yellowish oil; 1H NMR (CDCl3) δ 8.63 (brs), 8.40 (d, J = 5), 7.78 (d, J = 5), 7.53 (d, J = 2), 7.40 (dd, J = 8, 2), 6.40 (s), 5.91 (brs), 5.62 (q, J = 8.5), 5.52 (td, J = 10.5), 5.26 (t, J = 9), 4.31 (brs), 3.92(s), 3.50(d, J = 4), 3.08(m), 2.78 (m), 2.56 (m), and 1.2-2.5 (complex); 13C NMR (CDCl3) δ 154.2, 143.5, 139.9, 138.2, 137.4, 135.1, 134.5, 134.2, 132.3, 129.9, 129.2, 128.5, 122.2, 118.3, 117.2, 113.3, 112.5, 103.5, 75.1, 70.0, 68.7, 56.0, 55.0, 53.5, 50.9, 49.6, 47.1, 44.7, 40.9, 40.3, 32.7, 31.7, 28.1, 26.8, 26.0, 25.7, 21.7; HRESIMS m/z cacld for C37H47N4O2. [M+H]+ 579.3699, found 579.3704.
Methyl manzamine A-3-carboxylate (21b): 20d (26.9 mg) and DDQ (17.3 mg) were reacted as described above to gave 21b (18.1 mg, 68%). 21b: yellowish oil; 1H NMR (CDCl3) δ 9.25 (brs), 8.92 (s), 8.32 (d, J = 8), 7.77 (d, J = 8), 7.74 (t, J = 8), 7.50 (t, J = 8), 6.59 (s), 6.11 (br s), 5.79 (q, J = 8), 5.70 (td, J = 10.5), 5.44 (t, J = 9), 4.20 (s), 3.72 (brs), and 1.4-3.4 (complex); 13C NMR (CDCl3) δ 166.8, 143.4, 140.4, 139.5, 138.2, 137.2, 134.8, 132.3, 129.7, 129.3, 128.7, 128.4, 122.1, 121.6, 120.9, 116.5, 112.1, 74.9, 69.9, 68.6, 55.1, 53.6, 52.4, 50.9, 49.6, 47.2, 46.9, 44.8, 40.8, 40.5, 34.7, 33.1, 31.6, 28.2, 26.8, 25.9, 25.7, 21.6; HRESIMS m/z cacld for C38H47N4O3. [M+H]+ 607.3648, found 607.3655.
General Preparation of Compounds 17a-g
14 (54.8 mg, 0.1 mmol) was dissolved in 15 ml of chloroform and a catalytic amount of piperidine was added to the aromatic aldehyde (2.33 mg, 0.12 mmol) and stirred for an additional 5 minutes and then refluxed for 8 hr. The completion of the reaction was monitored by TLC and the chloroform in reaction mixture was evaporated and the residue was washed with water and extracted with chloroform. The crude product was chromatographed on a silica gel column using a hexane/acetone (7:3). The analogues of 14 (17a-g) were synthesized using the same reaction conditions.
31-(3-Methoxyphenyl)manzamine F (17a): yield 55%; 1H NMR (CDCl3) δ 8.21 (d, J = 5.2), 7.71 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.92 (s) 6.89 (d, J = 7.6), 6.39 (s), 5.02 (m), 5.40 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C44H51N4O4 [M+H]+ 699.3910, found 699.3888, [M+2H]++ 350.2013.
31-(4-Methoxyphenyl)manzamine F (17b): yield 50%; 1H NMR (CDCl3) δ 8.22 (d, J = 5.2), 7.71 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.92 (s) 6.89 (d, J = 7.6), 6.39 (s), 5.01 (m), 5.40 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); 13C NMR (CDCl3) δ 197.3, 159.3, 143.5, 143.3, 143.3, 140.2, 138.6, 138.2, 133.6, 132.3, 132.0, 131.8, 130.9, 129.7, 128.6, 123.4, 121.7, 121.2, 114.1, 113.6, 81.3, 76.2, 68.7, 63.6, 55.2, 53.1, 52.8, 49.7, 47.2, 46.6, 41.8, 41.5, 39.0, 33.3, 33.1, 26.7, 25.4, 24.9, 24.1, 21.8; HRESIMS m/z calcd for C44H50N4O4 [M+H]+ 699.3910, found 699.3887, [M+2H]++ 350.1977.
31-(4-Nitrophenyl)manzamine F (17c): yield 45 %; 1H NMR (CDCl3) δ 8.21 (d, J = 5.2), 7.7 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.92 (s) 6.89 (d, J = 7.6), 6.39 (s), 5.01 (m), 5.40 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C43H48N5O5 [M+H]+ 714.3655, found 714.3687, [M+2H]++ 357.6822.
31-(2,6-Dichlorophenyl)manzamine F(17d): yield 40%; 1H NMR (CDCl3) δ 8.20 (d, J = 5.2), 7.71 (d, J = 5.2), 7.52 (d, J = 7.6), 7.00 (t, J = 7.6), 6.92 (s) 6.89 (d, J = 7.6), 6.40 (s), 5.01 (m), 5.41 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.64 (s), 3.23 (brt, 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C43H47Cl2N4O3 [M+H]+ 737.3025, found 737.3028, [M+2H]++ 369.1557.
31-(3,4-Dimethoxyphenyl)manzamine F (17e): yield 47%; H NMR (CDCl3) δ 8.20 (d, J = 5.2), 7.71 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.91 (s) 6.89 (d, J = 7.6), 6.39 (s), 5.01 (m), 5.41 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C45H53N4O5 [M+H]+ 729.4016, found 729.4018, [M+2H]++ 365.2025.
31-(4-Bromophenyl)manzamine F (17f): yield 40 %; 1H NMR (CDCl3) δ 8.23 (d, J = 5.2), 7.72 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.94 (s), 6.89 (d, J = 7.6), 6.39 (s), 5.01 (m), 5.40 (ddd, J = 10.8, 10.8, 4), 3.91 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50(m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C43H48BrN4O3 [M+H]+ 747.2910, found 747.2930, [M+2H]++ 374.1516.
31-(4-Fluorophenyl)manzamine F (17g) :yield 45 %; 1H NMR (CDCl3) δ 8.21 (d, J = 5.2), 7.71 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.92 (s) 6.89 (d, J = 7.6), 6.39 (s), 5.01 (m), 5.40 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C43H48FN4O3 [M+H]+ 687.3710, found 687.3713, [M+2H]++ 344.1875.
31-(2-Chlorophenyl)manzamine F (17h): yield 45%; 1H NMR (CDCl3) δ 8.22 (d, J = 5.2), 7.73 (d, J = 5.2), 7.51 (d, J = 7.6), 7.02 (t, J = 7.6), 6.93 (s) 6.89 (d, J = 7.6), 6.39 (s), 5.03 (m), 5.42 (ddd, J = 10.8, 10.8, 4), 3.92 (brs), 3.82 (s), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); HRESIMS m/z calcd for C43H48ClN4O3 [M+H]+ 703.3415, found 703.3445, [M+2H]++ 352.1750.
Manzamine F-31-hydrazone (15): 14 (58 mg) 2 mL of anhydrous ethanol. Hydrazine hydrate (200 μL) was added and refluxed for 2 hours. The temperature was allowed to cool down to room temperature and 15 crystals formed. The mother liquid was purified using silica gel preparative TLC (CHCl3:MeOH 95:5) which gave additional amount of 15. 15; pale yellow crystals; 1H NMR δ 9.13 (s), 8.43 (d, J = 4.4), 8.09 (d, J = 7.6), 7.82 (d, J = 4.4), 7.53 (br), 7.25 (s), 6.42 (s), 5.90 (m), 5.62 (m), 5.52 (m), 5.26 (t, J = 9.6), 4.35 (br), 3.52 (s), 3.05 (m), 2.78 (m), 2.58 (m), 2.43∼1.86 (m), 1.80∼1.3 (m); HRESIMS m/z calcd for C36H47N6O2 [M+H]+ 595.3761, found 595.3782.
31β-Hydroxymanzamine F (16): 58mg of 14 was dissolved in 1 mL of methanol and cooled to 0°C. 20 mg of NaBH4 was added and stirred for 10 min. 10 mL of water was added and extracted with ether 5mL × 3. Ether was evaporated and the product was purified by silica gel preparative TLC (CHCl3:MeOH 9:1) to yield 16 (24 mg). 16: yellow powder; 1H NMR (CDCl3) δ 8.21 (d, J = 5.2), 7.71 (d, J = 5.2), 7.50 (d, J = 7.6), 7.00 (t, J = 7.6), 6.89 (d, J = 7.6), 6.39 (s), 5.01 (m), 5.40 (ddd, J = 10.8, 10.8, 4), 3.90 (brs), 3.64 (s), 3.23 (brt, J = 4.5), 2.84 (brs), 2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m), 1.77∼1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m); 13C NMR (CDCl3) δ 137.8, 136.7, 132.6, 127.6, 120.9, 113.8, 113.2, 112.5, 81.8, 70.3, 68.5, 63.2, 53.3, 52.6, 49.4, 48.0, 41.7, 40.4, 36.5, 35.5, 33.0, 30.8, 26.5, 25.5, 25.1, 24.9, 21.5; HRESIMS m/z calcd for C36H47N4O3 [M+H]+ 583.3648, found 583.3664.
31-Ethylmanzamine F (18): 14 (58 mg) was dissolved in 2 mL of tetrahydrofuran and cooled in dry ice acetone bath. 10 equivalents of 3M EtMgBr in ether was added. The solution was warmed up to room temperature and stirred for 24 hours. The mixture was poured into 5% NH4Cl under stirring and extracted using chloroform. The chloroform extract was purified using silica gel preparative TLC (CHCl3: MeOH 85:15) twice to yield 18 (18 mg). 18: yellow powder; 1H NMR (CDCl3) δ 11.15 (s), 10.1 (s), 8.80 (s), 8.35 (d, J = 4.8), 7.82 (d, J = 4.2), 7.59 (d, J = 7.6), 7.13 (t, J = 7.6), 7.05 (d, J = 7.6), 6.88 (s), 4.19 (s), 3.31 (t, J = 12.8), 2.98 (d, J = 12.8), 2.83 (m), 2.55 (m), 2.26∼2.40 (m), 2.16 (d, J = 11.6), 2.00 (t, J = 10.8), 1.80 (m), 1.53∼1.22 (m), 0.88 (t, J = 7.2); 13C NMR (DEPT 135) δ 137.9, 134.1, 132.8, 127.6, 121.1, 114.3, 114.2, 112.0, 82.4, 67.7, 64.4, 54.2, 53.2, 49.8, 45.2, 43.4, 40.5, 38.8, 37.1, 34.3, 33.5, 26.9, 25.5, 24.9, 24.6, 23.7, 21.3, 7.87; HRESIMS m/z calcd for C38H51N4O3 [M+H]+ 611.3961, found 611.3953.
In vitro Antimalarial Assay
Antimalarial activity was determined in vitro against chloroquine sensitive (D6, Sierra Leone) and resistant (W2, Indo China) strains of Plasmodium falciparum by measuring plasmodial LDH activity as described earlier.43,65 Test compounds were dissolved in DMSO (2 mg/ml). A 200 μL suspension of P. falciparum culture (2% parasitemia and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 μg/mL amikacin) was added to the wells of a 96-well plate containing 10 μL of serially diluted samples. The plate was flushed with a gas mixture of 90% N2, 5% O2, and 5% CO2 and incubated at 37 °C, for 72 h in a modular incubation chamber. Plasmodial LDH activity was determined by using Malstat™ reagent (Flow Inc., Portland, OR).66 Briefly, 20 μL of the incubation mixture was mixed with 100 μL of the Malstat™ reagent and incubated for 30 min. 20 μL of a 1:1 mixture of NBT/PES (Sigma, St. Louis, MO) was then added and the plate is further incubated for 1 h in dark. The reaction was stopped by adding 100 μL of a 5% acetic acid solution. The plate was read at 650 nm. Chloroquine and artemisinin were used as the positive controls. To determine the selectivity index of antimalarial activity of compounds, their in vitro cytotoxicity to mammalian cells (Vero cells) was also determined. Cells were seeded at a density of 25,000 cells/well and incubated for 24 h. Serially diluted samples were added and again incubated for 48 h. The number of viable cells was determined by Neutral Red assay.67 Doxorubicin was used as a positive control. IC50 values were obtained from dose response curves.
In Vitro Antimicrobial Assay
All organisms were obtained from the American Type Culture Collection (Manassas, VA) and include the fungi Candida albicans ATCC 90028 and Cryptococcus neoformans ATCC 90113, and the bacterium Mycobacterium intracellulare ATCC 23068. Susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) methods.68,69,70 M. intracellulare is tested using a modified method of Franzblau, et al..71 Samples were serially-diluted in 20% DMSO/saline and transferred in duplicate to 96 well flat bottom microplates. Microbial inocula were prepared by correcting the OD630 of microbe suspensions in incubation broth to afford final target inocula. Drug controls [Ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and Amphotericin B (ICN Biomedicals, Ohio) for fungi] were included in each assay. All organisms were read at either 630nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont) or 544ex/590em, (M. intracellulare) using the Polarstar Galaxy Plate Reader (BMG LabTechnologies, Germany) prior to and after incubation.
Anti-neuroinflammatory Assay
Rat neonatal microglia (2 × 105 cells) were seeded into each well of 24-well flat-bottom culture clusters and stimulated with Escherichia coli lipopolysaccharide (LPS) (0.3 ng/mL) in Dulbecco's modified Eagle medium + 10% fetal bovine serum + penicillin + streptomycin for 17 h in a humidified 5% CO2 incubator at 35.9 °C as described.34,13 Media were then removed, microglia washed with warm (37 °C) Hanks' balanced salt solution (HBSS) and then incubated with compounds(0.01-10 μM) or vehicle (DMSO) for 15 minutes prior to stimulation with phorbol 12-myristate 13-acetate (PMA) (1 μM). All experimental treatments were run in duplicate and in a final volume of 1 mL. Seventy minutes after PMA stimulation, HBSS was aspirated from each well and superoxide anion (O2-), thromboxane B2 (TXB2) and lactated dehydrogenase (LDH) release were determined as described.34,13 Table 3 shows data from 1-3 experiments and is expressed as the compound's inhibitory concentration 50% (IC50) for either O2- or TXB2. LDH release from microglia was determined spectrophotometrically as described.35 Microglia LDH release was expressed as the compound concentration (LDH50) that yielded 50% percent of LDH release observed after treatment of control microglia with Triton X-100 (0.1%).
Assay for Transport across BBB
MDR-MDCK cell line was grown in Dulbecco's Modified Eagles Medium as described earlier.72 For the transport experiment, cells with passage number 12-40 were seeded at a density of 75,000 cells/cm2 and grown for eight days in 12-well Transwell® plates to form a monolayer. The bidirectional transport assay was conducted in HBSS, pH 7.4, as the transport medium. Caffeine (5μM) was used as a standard drug for BBB transport. The transport of manzamine A free base and its salt (50 μM) was monitored in absorptive and secretory directions across MDR-MDCK monolayer for two hours. The concentration of manzamine A in the donor chamber as well as in the samples collected at different time intervals from the receiver chamber was determined by LC-MS method. The concentration of caffeine was determined by an HPLC method as reported previously.72 The cumulative amount of the transported drug was plotted against time to obtain the rate of transport (as shown in Fig 1) and the apparent permeability coefficient (as shown in Fig 2) was calculated from the equation:
with dq/dt = rate of transport, Co = initial concentration in the donor chamber, A= surface area of the filter
LC/MS Analytical
A stock solution of manzamine A was prepared by dissolving in DMSO. The standard solution was diluted with 4% DMSO, and 2% cyclodextrin in HBSS at level of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, and 5.0 μg/mL. Agilent HP1100 with Bruker micro-TOF was used. The analysis was performed on a C8 column (4.6×150 mm, 5μm, Phenomenex Luna) using a gradient of MeOH – 2mM NH4Ac (0.05% formic acid) from 40:60 to 80:20 over 8 minutes at a flow rate of 0.8 mL/min. A after column splitter at ratio of 1:5 was utilized. The mass spectrometer was operated in positive ESI mode. The nebulizer pressure was 2 Bar, source temperature was 200 degree, and drying gas was 4L/min. Extracted ion at m/z 549.4 was integrated and used for quantification. The standard curve was linear over the range of 0.05-5 μg/mL (R2=0.9975). The precision at level of 0.05, 0.5, 2.0 μg/mL were 3.85%, 3.21%, 2.85%, respectively. The recovery at level of 0.05, 0.5, 2.0 μg/mL were 109.0%, 94.71%, 101.2%, respectively. Samples collected from donor or receiver side of the monolayer were directly injected into LC/MS.
In vivo Antimalarial Assay
The in vivo antimalarial activity of 1 was determined in mice infected with Plasmodium berghei (NK-65 strain), originally obtained from the Walter Read Army Institute of Research, Silver Spring, MD. For multiple dose studies the four days Peter's suppressive test was modified to a three days treatment schedule as recommended by Medicines for Malaria Venture (www.mmv.org) for evaluation of suppressive and curative activities. Male mice (Swiss Webster strain) weighing 18-20 g were intraperitoneally inoculated with 2×107 parasitized red blood cells obtained from a highly infected donor mouse. Mice were divided into different groups with at least 5 mice in each group. 1 stock was prepared in SSV or 0.1 M HCl, as specified in respective studies. Further dilutions of 1 were prepared in SSV or 0.01 M HCl. The mice infected with P. berghei were orally administered 100 μl of the compound. The animals were closely observed for at least 2 hours after every dose for any apparent signs of toxicity. Blood smears were prepared on different days starting from 5 days post infection (through 28 days) by clipping the tail end, stained with Giemsa and the slides were observed under microscope for determination of parasitemia. Mice without parasitemia through day 28 post-infection were considered cured.
Docking studies
For the docking studies we used GOLD 3.1.1 software.45 The X-ray crystal structure of GSK-3β (pdb code: 1gng)47, 49 was retrieved from the Protein Data Bank. The co-crystallized sulfates were removed before the docking studies. For docking, the active site was defined as any atom that lay within a 15 Å radius of the δN of Arg96. The geometry of 2 was optimized using MMFF94 in Sybyl 7.2 (Tripos Asociates, St. Louis, Mo), starting from the published X-ray crystal structure of manzamine A.1 The top ranked binding pose was identified based on the GOLD fitness score and used for further analysis.
Supplementary Material
Acknowledgments
We gratefully acknowledge the National Institute of Health (1R01A136596 and 5K02A101502) and the Medicines for Malaria Venture (MMV) for financial support. The authors wish to thank Marsha Wright and John Trott for biological testing, which was supported by the NIH, NIAID, Division of AIDS, Grant No. AI 27094 and the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-6408-2-0009. Additional support to AMSM from Midwestern University and technical assistance by Ms. Mary Hall are gratefully acknowledged. RJD acknowledges funding from National Science Foundation (EPS-0556308), Centers for Disease Control and Prevention National Center for Zoonotic, Vector-Borne, and Enteric Diseases (NCZVED), American Association of Colleges of Pharmacy New Investigator Program, NIH National Center for Research Resources (NCRR) grant P20 RR021929 and C06 RR-14503-01, University of Mississippi's Office of Research and Sponsored Programs, Partners and Provost's Associates Grants. SP is a CORE-NPN Natural Products Neuroscience fellow.
Abbreviations
- TXB2
thromboxane B2
- BBB
blood-brain barrier
- Mtb
Mycobacterium tuberculosis
- DCM
dichloromethane
- P.O.
per os
- GSK-3β
glycogen synthesis kinase-3β
- HsGSK-3β
Homo sapiens GSK-3β
- PfGSK-3
Plasmodium falciparum parasite GSK-3
- AD
Alzheimer's disease
- LPS
lipopolysaccharide
- HBSS
Hanks' balanced salt solution
- PMA
phorbol 12-myristate 13-acetate
Footnotes
Supporting Information Available. Complete references 20 and 58, full list of Tables 1-3, and cytotoxicity studies. This material is available free of charge via the Internet at http://pubs.acs.org.
References
- 1.Sakai R, Higa T, Jefford CW, Bernardinelli G. Manzamine A, a novel antitumor alkaloid from a sponge. J Am Chem Soc. 1986;108:6404–6405. [Google Scholar]
- 2.Hu JF, Hamann MT, Hill R, Kelly M. The manzamine alkaloids. Alkaloids Chem Biol. 2003;60:207–285. doi: 10.1016/s0099-9598(03)60004-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Peng J, Rao KV, Choo YM, Hamann MT. In: Moden Alakloids. Fattorusso E, TS O, editors. Wiley-VCH Varlag Gmbh & Co.; KGaA: Weinheim Germany: 2008. pp. 189–232. [Google Scholar]
- 4.Rao KV, Santarsiero BD, Mesecar AD, Schinazi RF, Tekwani BL, Hamann MT. New manzamine alkaloids with activity against infectious and tropical parasitic diseases from an Indonesian sponge. J Nat Prod. 2003;66:823–828. doi: 10.1021/np020592u. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Hamann MT, El-Sayed KA. Methods of treating drug-resistant infections through administration of pharmaceutical compositions containing manzamine alkaloids. US27035. 2002
- 6.Nakamura H, Deng S, Kobayashi J, Ohizumi Y, Tomotake Y, Matsuzaki T, Hirata Y. Physiologically active marine natural products from Porifera. XV. Keramamine-A and -B, novel antimicrobial alkaloids from the Okinawan marine sponge Pellina sp. Tetrahedron Lett. 1987;28:621–624. [Google Scholar]
- 7.El Sayed KA, Kelly M, Kara UA, Ang KK, Katsuyama I, Dunbar DC, Khan AA, Hamann MT. New manzamine alkaloids with potent activity against infectious diseases. J Am Chem Soc. 2001;123:1804–1808. doi: 10.1021/ja002073o. [DOI] [PubMed] [Google Scholar]
- 8.Ang KK, Holmes MJ, Higa T, Hamann MT, Kara UA. In vivo antimalarial activity of the beta-carboline alkaloid manzamine A. Antimicrob Agents Chemother. 2000;44:1645–1649. doi: 10.1128/aac.44.6.1645-1649.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Longley RE, McConnell OJ, Essich E, Harmody D. Evaluation of marine sponge metabolites for cytotoxicity and signal transduction activity. J Nat Prod. 1993;56:915–920. doi: 10.1021/np50096a015. [DOI] [PubMed] [Google Scholar]
- 10.Mayer AMS, Gunasekera SP, Pomponi SA, Sennett SH. Anti-inflammatory uses of manzamine. US 20020187999. 2002
- 11.Mayer AMS, Gunasekera SP, Pomponi SA, Sennett SH. Anti-inflammatory uses of manzamines. US6,602,881. 2003
- 12.Yousaf M, Hammond NL, Peng J, Wahyuono S, McIntosh KA, Charman WN, Mayer AM, Hamann MT. New manzamine alkaloids from an Indo-Pacific sponge. Pharmacokinetics, oral availability, and the significant activity of several manzamines against HIV-I, AIDS opportunistic infections, and inflammatory diseases. J Med Chem. 2004;47:3512–3517. doi: 10.1021/jm030475b. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Mayer AM, Hall ML, Lynch SM, Gunasekera SP, Sennett SH, Pomponi SA. Differential modulation of microglia superoxide anion and thromboxane B2 generation by the marine manzamines. BMC Pharmacol. 2005;5:6. doi: 10.1186/1471-2210-5-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Peng J, Shen X, El Sayed KA, Dunbar DC, Perry TL, Wilkins SP, Hamann MT, Bobzin S, Huesing J, Camp R, Prinsen M, Krupa D, Wideman MA. Marine natural products as prototype agrochemical agents. J Agric Food Chem. 2003;51:2246–2252. doi: 10.1021/jf0207880. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Rao KV, Kasanah N, Wahyuono S, Tekwani BL, Schinazi RF, Hamann MT. Three new manzamine alkaloids from a common Indonesian sponge and their activity against infectious and tropical parasitic diseases. J Nat Prod. 2004;67:1314–1318. doi: 10.1021/np0400095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Peng J, Hu JF, Kazi AB, Li Z, Avery M, Peraud O, Hill RT, Franzblau SG, Zhang F, Schinazi RF, Wirtz SS, Tharnish P, Kelly M, Wahyuono S, Hamann MT. Manadomanzamines A and B: a novel alkaloid ring system with potent activity against mycobacteria and HIV-1. J Am Chem Soc. 2003;125:13382–13386. doi: 10.1021/ja030087z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Yousaf M, El Sayed KA, Rao KV, Lim CW, Hu JF, Kelly M, Franzblau SG, Zhang F, Peraud O, Hill RT, Hamann MT. 12,34-Oxamanzamines, novel biocatalytic and natural products from manzamine producing Indo-Pacific sponges. Tetrahedron. 2002;58:7397–7402. [Google Scholar]
- 18.El Sayed KA, Bartyzel P, Shen X, Perry TL, Zjawiony JK, Hamann MT. Marine natural products as antituberculosis agents. Tetrahedron. 2000;56:949–953. [Google Scholar]
- 19.Kondo K, Ohnishi N, Takemoto K, Yoshida H, Yoshida K. Synthesis of optically quadratic nonlinear phenylpyridylacetylenes. J Org Chem. 1992;57:1622–1625. [Google Scholar]
- 20.Hamann M, Alonso D, Martin-Aparicio E, Fuertes A, Perez-Puerto MJ, Castro A, Morales S, Navarro ML, Del Monte-Millan M, Medina M, Pennaka H, Balaiah A, Peng J, Cook J, Wahyuono S, Martinez A. Glycogen synthase kinase-3 (GSK-3) inhibitory activity and structure-activity relationship (SAR) studies of the manzamine alkaloids. Potential for Alzheimer's disease. J Nat Prod. 2007;70:1397–1405. doi: 10.1021/np060092r. [DOI] [PubMed] [Google Scholar]
- 21.Liu F, Liang Z, Shi J, Yin D, El-Akkad E, Grundke-Iqbal I, Iqbal K, Gong CX. PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners. FEBS Lett. 2006;580:6269–6274. doi: 10.1016/j.febslet.2006.10.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Winkler JD, Axten JM. The First Total Syntheses of Ircinol A, Ircinal A, and Manzamines A and D. J Am Chem Soc. 1998;120:6425–6426. doi: 10.1021/ja981303k. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Martin SF, Humphrey JM, Ali A, Hillier MC. Enantioselective Total Syntheses of Ircinal A and Related Manzamine Alkaloids. J Am Chem Soc. 1999;121:866–867. [Google Scholar]
- 24.Humphrey JM, Liao Y, Ali A, Rein T, Wong YL, Chen HJ, Courtney AK, Martin SF. Enantioselective total syntheses of manzamine a and related alkaloids. J Am Chem Soc. 2002;124:8584–8592. doi: 10.1021/ja0202964. [DOI] [PubMed] [Google Scholar]
- 25.Pandit UK, Overkleeft HS, Borer BC, Bieraugel H. Synthesis mediated by ring-closing metathesis. Applications in the synthesis of azasugars and alkaloids. Eur J Org Chem. 1999;(5):959–968. [Google Scholar]
- 26.Uchida H, Nishida A, Nakagawa M. An efficient access to the optically active manzamine tetracyclic ring system. Tetrahedron Lett. 1999;40:113–116. [Google Scholar]
- 27.Coldham I, Crapnell KM, Fernandez JC, Moseley JD, Rabot R. Synthesis of the ABC ring system of manzamine A. J Org Chem. 2002;67:6181–6187. doi: 10.1021/jo016376s. [DOI] [PubMed] [Google Scholar]
- 28.Baldwin JE, Claridge TDW, Culshaw AJ, Heupel FA, Lee V, Spring DR, Whitehead RC, Boughtflower RJ, Mutton IM, Upton RJ. Investigations into the manzamine alkaloid biosynthetic hypothesis. Angew Chem, Int Ed. 1998;37:2661–2663. doi: 10.1002/(SICI)1521-3773(19981016)37:19<2661::AID-ANIE2661>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
- 29.Winkler JD, Londregan AT, Ragains JR, Hamann MT. Synthesis and biological evaluation of manzamine analogues. Org Lett. 2006;8:3407–3409. doi: 10.1021/ol061320b. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Winkler JD, Londregan AT, Hamann MT. Antimalarial activity of a new family of analogues of manzamine A. Org Lett. 2006;8:2591–2594. doi: 10.1021/ol060848d. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Torisawa Y, Hashimoto A, Okouchi M, Iimori T, Nagasawa M, Hino T, Nakagawa M. Manzamine C congeners with modified azacyclic rings: synthesis and biological evaluation. Bioorg Med Chem Lett. 1996;6:2565–2570. [Google Scholar]
- 32.Winkler JD, Londregan AT, Hamann MT. Synthetic modification of manzamine A via Grubbs metathesis. Novel structures with enhanced antibacterial and antiprotozoal properties. Org Lett. 2007;9:4467–4469. doi: 10.1021/ol701799c. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Choo YM, Hamann MT. An improved Pictet-Spengler condensation: a convenient synthetic route to bioactive manzamine derivatives. Heterocycles. 2007;71:245–252. [Google Scholar]
- 34.Mayer AM, Oh S, Ramsey KH, Jacobson PB, Glaser KB, Romanic AM. Escherichia coli lipopolysaccharide potentiation and inhibition of rat neonatal microglia superoxide anion generation: correlation with prior lactic dehydrogenase, nitric oxide, tumor necrosis factor-alpha, thromboxane B2, and metalloprotease release. Shock(Augusta,Ga) 1999;11:180–186. [PubMed] [Google Scholar]
- 35.Morgenstern S, Flor R, Kessler G, Klein B. The automated determination of NAD-coupled enzymes. II. Serum lactate dehydrogenase. Clin Chem. 1966;12:274–281. [PubMed] [Google Scholar]
- 36.Deininger MH, Kremsner PG, Meyermann R, Schluesener H. Macrophages/microglial cells in patients with cerebral malaria. Eur Cytokine Network. 2002;13:173–185. [PubMed] [Google Scholar]
- 37.Van der Heyde HC, Nolan J, Combes V, Gramaglia I, Grau GE. A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction. Trends Parasitol. 2006;22:503–508. doi: 10.1016/j.pt.2006.09.002. [DOI] [PubMed] [Google Scholar]
- 38.Schluesener HJ, Kremsner PG, Meyermann R. Widespread expression of MRP8 and MRP14 in human cerebral malaria by microglial cells. Acta Neuropathol. 1998;96:575–580. doi: 10.1007/s004010050938. [DOI] [PubMed] [Google Scholar]
- 39.Woodgett JR. In: Glycogen synthase Kinase 3 (GSK-3) and its Inhibitors. Martinez A, Castro A, Medina M, editors. John Wiley & Sons,Inc.; Hoboken, N.J.: 2006. pp. 3–23. [Google Scholar]
- 40.Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cellular fate. Cell Mol Life Sci. 2007;64:1930–1944. doi: 10.1007/s00018-007-7045-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol. 2001;2:769–776. doi: 10.1038/35096075. [DOI] [PubMed] [Google Scholar]
- 42.Meijer L, Flajolet M, Greengard P. Pharmacological inhibitors of glycogen synthase kinase 3. Trends Pharmacol Sci. 2004;25:471–480. doi: 10.1016/j.tips.2004.07.006. [DOI] [PubMed] [Google Scholar]
- 43.Rao KV, Donia MS, Peng J, Garcia-Palomero E, Alonso D, Martinez A, Medina M, Franzblau SG, Tekwani BL, Khan SI, Wahyuono S, Willett KL, Hamann MT. Manzamine B and E and ircinal A related alkaloids from an Indonesian Acanthostrongylophora sponge and their activity against infectious, tropical parasitic, and Alzheimer's diseases. J Nat Prod. 2006;69:1034–1040. doi: 10.1021/np0601399. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Tu LC, Chou CK, Chen CY, Chang YT, Shen YC, Yeh SF. Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine alkaloids. Biochim Biophys Acta. 2004;1672:148–156. doi: 10.1016/j.bbagen.2004.03.007. [DOI] [PubMed] [Google Scholar]
- 45.Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD. Improved protein-ligand docking using GOLD. Proteins. 2003;52:609–623. doi: 10.1002/prot.10465. [DOI] [PubMed] [Google Scholar]
- 46.Dajani R, Fraser E, Roe SM, Young N, Good V, Dale TC, Pearl LH. Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell. 2001;105:721–732. doi: 10.1016/s0092-8674(01)00374-9. [DOI] [PubMed] [Google Scholar]
- 47.Bax B, Carter PS, Lewis C, Guy AR, Bridges A, Tanner R, Pettman G, Mannix C, Culbert AA, Brown MJ, Smith DG, Reith AD. The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation. Structure. 2001;9:1143–1152. doi: 10.1016/s0969-2126(01)00679-7. [DOI] [PubMed] [Google Scholar]
- 48.Martinez A, Alonso M, Castro A, Perez C, Moreno FJ. First non-ATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer's disease. J Med Chem. 2002;45:1292–1299. doi: 10.1021/jm011020u. [DOI] [PubMed] [Google Scholar]
- 49.Martinez A, Alonso M, Castro A, Dorronsoro I, Gelpi JL, Luque FJ, Perez C, Moreno FJ. SAR and 3D-QSAR studies on thiadiazolidinone derivatives: exploration of structural requirements for glycogen synthase kinase 3 inhibitors. J Med Chem. 2005;48:7103–7112. doi: 10.1021/jm040895g. [DOI] [PubMed] [Google Scholar]
- 50.Droucheau E, Primot A, Thomas V, Mattei D, Knockaert M, Richardson C, Sallicandro P, Alano P, Jafarshad A, Baratte B, Kunick C, Parzy D, Pearl L, Doerig C, Meijer L. Plasmodium falciparum glycogen synthase kinase-3: molecular model, expression, intracellular localization and selective inhibitors. Biochim Biophys Acta, Proteins Proteomics. 2004;1697:181–196. doi: 10.1016/j.bbapap.2003.11.023. [DOI] [PubMed] [Google Scholar]
- 51.Martin M, Rehani K, Jope RS, Michalek SM. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol. 2005;6:777–784. doi: 10.1038/ni1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Jope RS, Yuskaitis CJ, Beurel E. Glycogen synthase kinase-3 (GSK3): inflammation, diseases, and therapeutics. Neurochem Res. 2007;32:577–595. doi: 10.1007/s11064-006-9128-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Magae Y, Magae J. Effect of staurosporine on growth and hyphal morphology of Pleurotus ostreatus. J Gen Microbiol. 1993;139:161–164. [Google Scholar]
- 54.Park HJ, Lee JY, Hwang IS, Yun BS, Kim BS, Hwang BK. Isolation and antifungal and antioomycete activities of staurosporine from Streptomyces roseoflavus strain LS-A24. J Agric Food Chem. 2006;54:3041–3046. doi: 10.1021/jf0532617. [DOI] [PubMed] [Google Scholar]
- 55.Hunt NH, Golenser J, Chan-Ling T, Parekh S, Rae C, Potter S, Medana IM, Miu J, Ball HJ. Immunopathogenesis of cerebral malaria. Int J Parasitol. 2006;36:569–582. doi: 10.1016/j.ijpara.2006.02.016. [DOI] [PubMed] [Google Scholar]
- 56.Szklarczyk A, Stins M, Milward EA, Ryu H, Fitzsimmons C, Sullivan D, Conant K. Glial activation and matrix metalloproteinase release in cerebral malaria. J Neurovirol. 2007;13:2–10. doi: 10.1080/13550280701258084. [DOI] [PubMed] [Google Scholar]
- 57.Medana IM, Hunt NH, Chan-Ling T. Early activation of microglia in the pathogenesis of fatal murine cerebral malaria. Glia. 1997;19:91–103. doi: 10.1002/(sici)1098-1136(199702)19:2<91::aid-glia1>3.0.co;2-c. [DOI] [PubMed] [Google Scholar]
- 58.Hiestand PC, Petersen F, Roggo S, Hamann MT. Use of manzamines in transplantation and autoimmune diseases. 2006 EP7678. [Google Scholar]
- 59.Golenser J, McQuillan J, Hee L, Mitchell AJ, Hunt NH. Conventional and experimental treatment of cerebral malaria. Int J Parasitol. 2006;36:583–593. doi: 10.1016/j.ijpara.2006.02.009. [DOI] [PubMed] [Google Scholar]
- 60.Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WS, Hampel H, Hull M, Landreth G, Lue L, Mrak R, Mackenzie IR, McGeer PL, O'Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R, Walker D, Webster S, Wegrzyniak B, Wenk G, Wyss-Coray T. Inflammation and Alzheimer's disease. Neurobiol Aging. 2000;21:383–421. doi: 10.1016/s0197-4580(00)00124-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61.Block ML, Hong JS. Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol. 2005;76:77–98. doi: 10.1016/j.pneurobio.2005.06.004. [DOI] [PubMed] [Google Scholar]
- 62.Sastre M, Klockgether T, Heneka MT. Contribution of inflammatory processes to Alzheimer's disease: molecular mechanisms. Int J Dev Neurosci. 2006;24:167–176. doi: 10.1016/j.ijdevneu.2005.11.014. [DOI] [PubMed] [Google Scholar]
- 63.Hill RT, Hamann MT, Peraud O, Kasanah N. Manzamine-producing actinomycetes. US24238. 2004
- 64.Shilabin AG, Kasanah N, Tekwani BL, Hamann MT. Kinetic studies and bioactivity of potential manzamine prodrugs. J Nat Prod. 2008;71:1218–1221. doi: 10.1021/np800163u. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65.Ibrahim MA, Shilabin AG, Prasanna S, Jacob M, Khan SI, Doerksen RJ, Hamann MT. 2-N-Methyl modifications and SAR studies of manzamine A. Bioorg Med Chem. 2008;16:6702–6706. doi: 10.1016/j.bmc.2008.05.079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, Hinrichs DJ. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg. 1993;48:739–741. doi: 10.4269/ajtmh.1993.48.739. [DOI] [PubMed] [Google Scholar]
- 67.Borenfreund E, Babich H, Martin-Alguacil N. Rapid chemosensitivity assay with human normal and tumor cells in vitro. In Vitro Cell Dev Biol. 1990;26:1030–1034. doi: 10.1007/BF02624436. [DOI] [PubMed] [Google Scholar]
- 68.NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of yeasts; Approved Standard M27-A2. 15 Vol. 22. National Committee on Clinical laboratory Standards; 2002. [Google Scholar]
- 69.NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of yeasts; Approved Standard M24-T2. 26 Vol. 20. National Committee on Clinical laboratory Standards; 2000. [Google Scholar]
- 70.NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically M7-A5. 2 Vol. 20. National committee on clinical laboratory standards; 2000. [Google Scholar]
- 71.Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM, Gilman RH. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol. 1998;36:362–366. doi: 10.1128/jcm.36.2.362-366.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 72.Madgula VL, Avula B, Reddy VLN, Khan IA, Khan SI. Transport of decursin and decursinol angelate across Caco-2 and MDR-MDCK cell monolayers: in vitro models for intestinal and blood-brain barrier permeability. Planta Med. 2007;73:330–335. doi: 10.1055/s-2007-967137. [DOI] [PubMed] [Google Scholar]
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