European Journal of Medicinal Chemistry 81 (2014) 15e21
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Identification of a neuroprotective and selective butyrylcholinesterase inhibitor derived from the natural alkaloid evodiamine Guozheng Huang a, Beata Kling b, Fouad H. Darras b, Jörg Heilmann b, Michael Decker a, * a b
Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2013 Received in revised form 7 April 2014 Accepted 1 May 2014 Available online 4 May 2014
Two sets of carbamates based on the natural alkaloid evodiamine were designed, synthesized and evaluated as potential butyrylcholinesterase inhibitors. Although a set of carbamates of 3hydroxyevodiamine (10aef) is inactive both at AChE and BChE, carbamates of 5-deoxo-3hydroxyevodiamine (11aef) exhibit much better potency with selectivity toward BChE. The heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c) shows the best potency with an IC50 value of 77 nM and very good selectivity over AChE. ORAC and cell-based assays indicate 11c owns pronounced antioxidant properties with 1.75 Trolox equivalents and strong neuroprotection even from 1 mM onwards. These combined activities might enable compound 11c to be a potential candidate for treatment of Alzheimer’s disease. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Alzheimer’s disease Butyrylcholinesterase inhibitor Neuroprotection Evodiamine Carbamates
1. Introduction Alzheimer’s disease (AD), the most prominent form of dementia, is clinically defined as a progressive and irreversible loss of cognitive functions combined with neuropsychiatric disturbance. Although numerous efforts have been dedicated to the investigation of its etiologies, no effective method is yet available to prevent or reverse AD and its pathogenesis still remains unclear [1]. Several hypotheses have been advocated to constitute the key pathological hallmarks of AD, including b-amyloid deposits, s-protein aggregation, oxidative stress, and cholinergic system dysfunction [2]. According to the cholinergic hypothesis, drastic deficit of the neurotransmitter acetylcholine is the ultimate reason for failure in neurotransmission and subsequent aggravating cognitive and memory symptoms. Therefore, acetylcholinesterase (AChE) inhibitors are utilized clinically for the treatment of mild AD, such as tacrine, donepezil, rivastigmine and galantamine [3]. Current medicinal chemistry research focuses on novel AChE inhibitors, often with multifunctional and multi-target activities [4,5]. The second type of cholinesterase in the human body, butyrylcholinesterase (BChE), is also known as plasma cholinesterase. Although its portfolio of functions is still not completely
* Corresponding author. E-mail address: [email protected] (M. Decker). http://dx.doi.org/10.1016/j.ejmech.2014.05.002 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
understood, it may be also involved in the normal nervous system function and might participate in pathological processes in the etiology of AD as elucidated in several studies [6]. During later stage of AD, the activity of AChE decreases to reach 10e15% of normal values in certain brain regions, while the amount of BChE remains the same or even increases up to 2-fold [7]. Hence, selective AChE inhibitors might therefore encounter clinical ineffectiveness in later stages of AD. Actually, a balance between AChE and BChE inhibition or selectivity toward BChE might be more desirable. Furthermore, in vivo studies proved selective BChE inhibitors elevate brain acetylcholine, lower b-amyloid peptide, and ameliorate cognitive dysfunction [8,9]. In summary, selective BChE inhibitors could be of importance for treatment of AD and exploration of the pathogenesis of AD. Up to now, only a few selective BChE inhibitors (with selectivity index, SI > 100) were developed, such as heterobivalent tacrine derivatives [10], benzofurans [11] and isosorbide-based compounds [12] (the most potent one is isosorbide-2-benzyl carbamate-5salicylate, 1), cymserine analogs [13,14] (N1-phenylethyl-norcyserine, 2), and diarylimidazole compounds [15] (3). Our group used quinazolinimines as lead structures to develop a series of Nbridgeheaded tri- and tetracylic compounds with improved selectivity toward BChE [16e18]. Further exploration on the related structures identified a carbamate-based tetracyclic lead structure (4) [19]. Performing SARs on a library of tri- and tetracyclic structures with and without an additional carbamate moiety revealed
16
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
that a tetracyclic structure holds better potency and selectivity than the tricyclic structure. This result intrigued us to explore the possibility of better interaction between the aromatic ring and the binding region of BChE. It is well established that oxidative stress, the formation of reactive oxygen species (ROS), and subsequent neurotoxicity are key processes in the pathophysiology of AD [20]. We showed previously that compound 4 and its analogs possess very significant antioxidant properties (determined both with regard to their physico-chemical radical scavenging properties as well as with regard to neuron protection against oxidative stress) [19]. Evodiamine is the major quinazoline alkaloid isolated from Evodia rutaecarpa used in Traditional Chinese Medicine for treatment of a variety of ailments including headache, abdominal pain, postpartum hemorrhage, dysentery and amenorrhea [21,22]. Several research efforts indicate that evodiamine and its chemical derivatives are potent anti-cancer drug candidates [23,24]. The physiological effects of evodiamine on aged animals implicate a potential for the treatment of erectile dysfunction [25]. An in vivo study proved that it also can improve cognitive abilities in a transgenic mouse model of AD [26]. Recently, Lee et al. described a specific inhibitory activity on BChE with an IC50 value of 1.7 mg/mL (corresponding to 5.6 mM) although this value is far lower than for the compounds shown in Chart 1 [27]. In this study, we wanted to extend and continue our earlier studies seeking highly selective and potent BChE inhibitors with pronounced neuroprotective effects by combining the potent natural compound evodiamine with a privileged carbamate scaffold and applying the knowhow we had gained from previous tetracyclic scaffolds. 2. Results and discussion 2.1. Chemistry Condensation of 4,9-dihydro-3H-pyrido[3,4-b]indole (6) and 6(benzyloxy)-1-methyl-1H-benzo[d] [1,3]oxazine-2,4-dione (5) in refluxing dichloromethane afforded the desired 3benzyloxyevodiamine (7) in excellent yield (97%) (Scheme 1).
Fig. 2. Evaluation of neurotoxicity (A) and neuroprotection (B) of compounds 14, 9, 10c and 11c, respectively, against glutamate induced oxidative stress on HT-22 cells. Results are presented as means SD and refer to untreated control cells which were set as 100% values. Experiments were carried out with 4 parallels and repeated independently at least three times. Data were subjected to one-way ANOVA followed by Dunnet’s multiple comparison post-test using GraphPad Prism 5 Software. Levels of significance are: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
Intermediate 6 could be obtained from indole via a six-step linear synthesis. The isatoic anhydride derivative 5, which represents an activated form of anthranilic acid with a benzylated phenolic OH, was synthesized in five steps with almost quantitative overall yield starting from 5-hydroxyanthranilic acid (cf. Supplementary Material for the syntheses of the two intermediates). Benzylation of the phenolic group in compound 5 is necessary to avoid methylation of the phenolic group and to improve solubility in dichloromethane for subsequent fusion reaction. 3-Benzyloxyevodiamine (7) was
Fig. 1. (A) Time-dependent pattern of BChE inhibition by compound 11c (0.25e1 mM) and (B) Stability constants of the inhibitoreBChE complex (KC) and rate constants of carbamoyl-ChE formation (k3) of 11c and physostigmine, respectively.
Chart 1. Structures of selective BChE inhibitors.
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
17
Scheme 1. Synthetic protocol for target carbamates 10aef and 11aef. Reagents and conditions: (i) CH2Cl2, reflux, 20 h; (ii) 1 atm H2, THF, r.t., 20 h; (iii) LiAlH4, THF, r.t., 8 h; (iv) isocyanate, Hünig’s base, THF, 2e4 days; (v) isocyanate, triethylamine, CH2Cl2, 2e4 h.
debenzylated by hydrogenation to convert it into 3hydroxyevodiamine (8). Carbamoylation of 3-hydroxyevodiamine with appropriate isocyanates in the presence of Hünig’s base generated the target carbamates 10aef [19]. 3-Hydoxyevodiamine was reduced by lithium aluminum hydride to give 5-deoxo-3hydroxyevodiamine (9), which could also be carbamoylated using isocyanates in the presence of triethylamine to afford the second type of carbamates (11aef) (Scheme 1). Evodiamine (13) and 5deoxoevodiamine (14) were synthesized according to a recent literature method [23] (Scheme 2). The compounds’ structures and purities were fully supported by analytical and spectral data (1H NMR, 13C NMR, HPLC, and HRMS). 2.2. Pharmacology 2.2.1. Inhibition assay of AChE and BChE Synthesized carbamates and synthetic intermediates, together with evodiamine and 5-deoxoevodiamine, were investigated for inhibition of the hydrolase activity of AChE and BChE using Ellman’s reaction [19,28]. The results are summarized in Table 1. Our lead structure evodiamine (13) is moderately potent toward BChE with an IC50 of 4.62 mM and shows moderate selectivity, which is consistent with literature [27]. Reduction of evodiamine to 14 resulted in higher potency and better selectivity which indicates removal of 5-position’s amide oxygen is well tolerated for maintaining potency. In contrast to our expectation, introduction of 3hydroxy group into evodiamine rendered compound 8 to lose activity on both AChE and BChE. This is in strong contrast to the
respective benzo analog we previously described [19]. Nevertheless, reduction of 8 to 5-deoxo-3-hydroxyevodiamine 9 recovered potency, with moderate selectivity toward BChE. Surprisingly, carbamates of 3-hydroxyevodiamine (10aef) are not inhibiting either AChE or BChE at 10 mM, while after carbamoylation, the set of 5-deoxo-3-hydroxyevodiamine derivatives (11aef) are exhibiting better potency than 5-deoxo-3hydroxyevodiamine 9. Aromatic carbamates (11def) are less active with IC50 values higher than 10 mM (inhibition at higher concentrations could not be determined because of insolubility at >10 mM). Aliphatic carbamates (11aec) are more potent though, showing IC50 values in the micromolar and submicromolar range. The by far most potent and most selective BChE inhibitor is the heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c), which represents a 2-digit nanomolar inhibitor with no significant activity on AChE even at 10 mM. Its potency toward BChE was as good as the positive control physostigmine, but exhibiting pronounced BChE selectivity. 2.2.2. Kinetic study of BChE inhibition Rivastigmine (ExelonÒ) and the natural product physostigmine represent pseudo-irreversible inhibitors transferring the carbamate moiety to the serine unit in the active center of AChE and BChE (pseudo-irreversible, since the carbamate subsequently slowly hydrolyzes off the enzyme). Based on the structural similarity of 3-hydroxyevodiamine 11c and physostigmine, we assumed the 11c would own similar pseudo-irreversible inhibitory behavior. In order to obtain more detailed information about the time course
Scheme 2. Synthetic protocol for evodiamine (13) and 5-deoxoevodiamine (14) synthesis. Reagents and conditions: (i) CH2Cl2, reflux, 20 h; (ii) LiAlH4, THF, r.t., 8 h.
18
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
Table 1 AChE and BChE inhibition, selectivity index and antioxidant capacities expressed as Trolox equivalents (concentration range 1e5 mM). Test compound
IC50, mM (pIC50 SEM or inhibition in % at 10 mM) AChEa
Physostigmine 3-Benzyloxyevodiamine (7) 3-Hydroxyevodiamine (8) 5-Deoxo-3-hydroxyevodiamine (9) R ¼ ethyl (10a) R ¼ butyl (10b) R ¼ heptyl (10c)
(10d)
0.0077 3.01 >100 >100 >10 >10 >10
SIb
Trolox equ.
BChEa (8.11 0.08) (5.52 0.06) (29%) (43% at 100 mM) (15%) (10%) (13%)
0.060 0.880 >100 19.3 >10 >10 >10
(7.22 0.06) (6.07 0.05) (47% at 100 mM) (4.71 0.05) (21%) (12%) (1%)
>10 (10%)
>10 (15%)
>10 (14%)
>10 (7%)
>10 (15%)
>10 (3%)
0.13 3.42 >5.17
3.55 0.03
0.32 0.03
(10e)
(10f) R ¼ ethyl (11a) R ¼ butyl (11b) R ¼ heptyl (11c)
(11d)
>10 (26%) >10 (47%) >10 (17%)
1.03 (5.99 0.06) 3.97 (5.40 0.12) 0.077 (7.11 0.06
>9.71 >2.52 >>130
>10 (34%)
2.15 (5.67 0.10)
>4.65
>10 (13%)
>10 (41%)
>10 (19%)
>10 (30%)
>10 (37%) >10 (24%)
4.62 (5.34 0.10) 2.68 (5.57 0.09)
1.75 0.14
(11e)
(11f) Evodiamine (13) 5-Deoxoevodiamine (14) a b
>2.16 >3.73
2.32 0.24
AChE from electric eel and BChE from equine serum; data are the means of at least 3 independent determinations. Selectivity index [IC50(AChE)/IC50(BChE)].
of inhibition and therefore more precise data about the equilibrium constant of inhibition, a kinetic study of compound 11c (and physostigmine as positive control) was performed on BChE (Fig. 1) [19]. Compound 11c shows a time-dependent pattern of inhibition characterized by an increase until a steady state after 60 min. The equilibrium constant of the inhibitoreBChE complex (KC) and the carbamoylation rate constant (k3) of 11c were determined (Fig. 1). The heptyl carbamate 11c represents a highly potent pseudoirreversible BChE inhibitor with KC ¼ 492 nM. The rate constant of 0.243 min1 shows rapid carbamoylation.
carbamate transfer to the enzyme; therefore this potent antioxidant is the neuroprotectant to act in vivo. Surprisingly, 5deoxoevodiamine 14, without a free hydroxy group is also potent with 2.32 Trolox equivalents. Finally, carbamate 10c is almost inactive in the ORAC assay with only 0.32 Trolox equivalents (Table 1). All the indolo compounds exhibit significantly higher Trolox values compared to the previously described benzo compounds, this is especially true for the unsubstituted compound 5deoxoevodiamine 14, which shows the importance of the indole ring with regard to antioxidant properties [19].
2.2.3. Antioxidant capacities (ORAC assay) For evaluation of antioxidant potencies, in a first assay the target compounds’ radical scavenging capacities were determined by means of their ability to reduce the amount of peroxyl radicals (oxygen radical absorbance capacity assay, ORAC assay). The most potent compound 11c and its free hydroxy group containing starting material 9 were chosen. Also 5-deoxoevodiamine (14) was tested for comparison to screen the influence of the free hydroxy group. Compound 10c was tested to evaluate the influence of the 5position amide’s oxygen. As expected, 11c is potent with antioxidant capacity of 1.75 Trolox equivalents, meaning that it is more potent than the positive control Trolox, a water-soluble vitamin E derivative. 5-Deoxo-3-hydroxyevodiamine 9 with a free hydroxy group is much more potent with 3.55 Trolox equivalents, which indicates a free hydroxy group is favorable for antioxidant potency. The hydroxyl compound 9 is formed from compound 11c after
2.2.4. Neuroprotectivity To obtain physiologically relevant data of antioxidant compounds with regard to neuroprotection, the neuronal cell line HT22 was chosen as a model system, which is a glutamate sensitive cell line and derived from murine hippocampal tissue [29]. As these cells lack ionotropic glutamate receptors, addition of extracellular glutamate in high concentrations inhibits cystine (as oxidized form of cysteine) transport via the cystine/glutamate antiporter. This neuronal oxidative stress induced toxicity leads to intracellular cysteine and therefore glutathione depletion which induces intracellular ROS accumulation and cell injuries [30e32]. The same compounds selected for the ORAC assay (9, 10c, 11c, and 14) were subsequently evaluated at different concentrations ranging from 1 to 25 mM for their neuroprotective potential toward neuronal HT-22 cells exposed to glutamate and subsequent intracellular ROS generation and oxidative stress. Furthermore, the
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
compounds were tested for putative neurotoxic effects toward the HT-22 neurons to elucidate possible self-toxic effects toward the cells. Concerning potential neurotoxicity, neither compound 9 with its free hydroxy group nor the corresponding carbamate 11c significantly reduce the cells’ viability at concentrations 1, 5 or 10 mM but significantly reduce the viability of HT-22 neurons at 25 mM each (Fig. 2A). The same observation can be made for 5deoxoevodiamine 14. In contrast to these compounds, the carbamate 10c reveals significant neurotoxic effects by reducing the cells’ viability at all tested concentrations from 1 mM onward e this compound was inactive in the ORAC assay. As expected, compound 9 with the free hydroxy group that is also formed after BChE carbamoylation from compound 11c shows prominent neuroprotection toward the glutamate challenged HT22 cells nearly to the same extent as the very potent positive control quercetin from 5 mM onwards (Fig. 2B). The protective effect is slightly reduced with increasing concentrations, probably due to the neurotoxic effect which was measurable from 25 mM onwards (Fig. 2A). Again, also 5-deoxoevodiamine 14 showed neuroprotective effects at 10 and 25 mM. Therefore, the physico-chemical radical scavenging properties determined in the ORAC assay are reflected in the neuronal cell-based assay. It has been shown before that not only compounds with the free hydroxy group but also the corresponding carbamates can act as neuroprotectants in their own regards [19]. And indeed evaluation of the corresponding heptyl carbamate (11c) of compound 9 revealed strong neuroprotective properties even from 1 mM onwards to the same extent as compound 9 (Fig. 2B). Additionally, the heptyl carbamate 10c, which varies only in one oxygen atom from compound 11c, still owns slightly measurable neuroprotective properties, which might even have been more pronounced without the compounds self-toxicity toward the cells that was detected and is shown in Fig. 2A. In summary, with regard to biological activities, both ORAC and hippocampal neuronal cell-based assays indicate that the most potent BChE inhibitor 11c also acts as a potent neuroprotectant. It is worth to note that although the free hydroxy group is favorable for neuroprotectivity, an indole ring system containing molecules without free hydroxy group also remains active in its own regard.
19
High-resolution ESI mass (HRMS) spectral data were acquired on an Agilent 1100 series LC/MSD ion trap mass spectrometer and a Agilent 6520B UPLC-TOF-MS instrument (Agilent, Santa Clara, U.S.A.), respectively. Analytic HPLC was performed on a system from Shimadzu Products equipped with a DGU-20A3R controller, LC20AB liquid chromatograph, and a SPD-20A UV/Vis detector. Stationary phase was a Synergi 4U fusion-RP (150 4.6 mm) column. As mobile phase, a gradient MeOH-TFA (0.01%)/water-TFA (0.01%) (phase A/ phase B) were used. Gradient mode: 0e8 min (30e90% phase A), 8e 13 min (90% phase A), 15e18 min (90e30% phase A). Intermediates 5, 6, 12, evodiamine (13), and 5-deoxoevodiamine (14), respectively, were synthesized according to literature procedures describing individual steps, but not in the required sequence. Therefore, sequence and synthetic details can be found in the Supplementary Material. 4.1. Synthesis of 3-(benzyloxy)-14-methyl-7,8,13b,14tetrahydroindolo[20 ,30 :3,4] pyrido[2,1-b]quinazolin-5(13H)-one (3benzyloxyevodiamine, 7) A solution of 6-(benzyloxy)-1-methyl-1H-benzo[d][1,3] oxazine-2,4-dione (5, 1.416 g, 5 mmol) and 4,9-dihydro-3H-pyrido[3,4b]indole (6, 0.87 g, 5.11 mmol, 1.02 eq.) in CH2Cl2 (30 mL) was stirred under reflux for 20 h. Then it was concentrated, the residue was rinsed in diethyl ether to give the product as a yellow solid (1.93 g, 97%). 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, NH), 7.71 (d, J ¼ 2.7 Hz, 1H), 7.63e7.57 (m, 1H), 7.49e7.37 (m, 5H), 7.36e7.31 (m, 1H), 7.29e7.24 (m, 1H), 7.21e7.07 (m, 3H), 5.88 (t, J ¼ 1.4 Hz, 1H, C13b-H), 5.16e5.06 (m, 2H, PhCH2O), 4.91e4.84 (m, 1H, C7eH), 3.34e3.26 (m, 1H, C7eH), 3.03e2.88 (m, 2H, C8eH), 2.38 (s, 3H, NCH3). 13C NMR (101 MHz, CDCl3): d 164.60 (C5), 155.96, 144.72, 136.89, 136.87, 128.76 (2 C), 128.40, 128.21, 127.76 (2 C), 126.41, 125.18, 124.78, 123.19, 122.09, 120.17, 119.06, 113.68, 112.22, 111.48, 70.62 (PhCH2O), 69.22 (C13b), 39.55 (C7), 37.52 (NCH3), 20.31 (C8). HPLC purity ¼ 98.65%, tR ¼ 9.58 min. HRMS (ESI): calcd for [M þ H]þ (C26H24N3O2) requires m/z 410.1863, found 410.1867. 4.2. Synthesis of 3-hydroxy-14-methyl-7,8,13b,14-tetrahydroindolo [20 ,30 :3,4] pyrido[2,1-b]quinazolin-5(13H)-one (3hydroxyevodiamine, 8)
3. Conclusion By derivation of the biologically active natural alkaloid evodiamine two sets of carbamates were identified with BChE inhibitory properties. The heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c) showed the best potency with an IC50 value of 77 nM and excellent selectivity over BChE, representing one of the few compounds with such an activity and selectivity profile. Carbamate 11c and hydroxy compound 9 formed after carbamate transfer are very potent neuroprotectants. 4. Experimental Common reagents and solvents were obtained from commercial suppliers and used without further purification. Tetrahydrofuran (THF) was distilled from sodium/benzophenone under argon atmosphere. Reactions were conducted using dried flasks under an atmosphere of nitrogen. Reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254 plates (MachereyeNagel GmbH & Co. KG, Düren, Germany) and spots were detected under UV light (254 nm). Nuclear magnetic resonance spectra were recorded with a Bruker AV-400 NMR instrument (Bruker, Karlsruhe, Germany) in DMSO-d6 or CDCl3. Chemical shifts are expressed in ppm relative to CDCl3 or DMSO-d6 (7.26/2.50 and 77.16/39.52 ppm for 1H and 13C NMR, respectively).
3-Benzyloxyevodiamine (7, 1.85 g, 4.518 mmol) in dried THF (100 mL) was hydrogenated under 1 atm of hydrogen catalyzed by 500 mg palladium on carbon (10%) at r.t. for 20 h. The catalyst was filtered off, the filtrate was concentrated to afford the 3hydroxyevodiamine as a yellow solid (1.39 g, 97%). 1H NMR (400 MHz, DMSO-d6): d 11.24 (s, 1H, NH), 9.46 (s, 1H, 3-OH), 7.51 (d, J ¼ 7.8 Hz,1H), 7.37 (d, J ¼ 8.1 Hz,1H), 7.29 (d, J ¼ 2.9 Hz,1H), 7.15e7.09 (m, 1H), 7.07 (d, J ¼ 8.6 Hz, 1H), 7.05e7.00 (m, 1H), 6.96 (dd, J ¼ 8.6, 2.9 Hz, 1H), 5.95 (s, 1H, C13b-H), 4.70e4.56 (m, 1H, C7eH), 3.14e3.18 (m, 1H, C7eH), 2.93e2.86 (m, 1H), 2.85e2.75 (m, 1H, C8eH), 2.37 (s, 3H, N14eCH3). 13C NMR (101 MHz, DMSO-d6): d 163.66 (C5), 153.38, 142.58, 136.83, 129.36, 125.73, 123.75, 123.50, 121.87, 120.85, 118.81, 118.36, 113.08, 111.61, 111.57, 68.95 (C13b), 39.38 (C7), 36.95 (NCH3), 19.80 (C8). HPLC purity ¼ 99.71%, tR ¼ 7.73 min. HRMS (ESI): calcd for [M þ H]þ (C19H18N3O2) requires m/z 320.1394, found 320.1396. 4.3. Synthesis of 14-methyl-5,7,8,13,13b,14-hexahydroindolo [20 ,30 :3,4]pyrido[2,1-b] quinazolin-3-ol(3-hydroxy-5deoxoevodiamine, 9) To the cooled suspension of lithium aluminum hydride (0.61 g, 16 mmol, 4 eq.) in dried THF (40 mL) was added a solution of 3hydroxyevodiamine (8, 1.29 g, 4 mmol) in dried THF (40 mL) dropwise at 0 C. After addition, the mixture was stirred at r.t. for
20
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
8 h, and then cooled on an ice-bath again. To the cooled mixture was added NaSO4$10H2O in small portions until no gas was liberated any more. Then it was filtered through a pad of Celite, the cake washed with CH2Cl2/MeOH (5/1, 5 40 mL). The filtrate was combined and concentrated in vacuo to give a yellow oil which was purified by column chromatography (CH2Cl2/MeOH ¼ 10/1) to afford the product as a yellow solid (1.05 g, 86%). 1H NMR (400 MHz, DMSO-d6): d 11.06 (s, 1H, N13eH), 8.98 (s, 1H, 3-OH), 7.44 (d, J ¼ 7.8 Hz, 1H), 7.33 (d, J ¼ 8.0 Hz, 1H), 7.10e7.02 (m, 1H), 7.01e6.94 (m, 1H), 6.88 (d, J ¼ 8.7 Hz, 1H), 6.60 (dd, J ¼ 8.6, 2.8 Hz, 1H), 6.47 (d, J ¼ 2.7 Hz, 1H), 4.64 (s, 1H, C13b-H), 3.93 (d, J ¼ 15.3 Hz, 1H, C5eH), 3.69 (d, J ¼ 15.2 Hz, 1H, C5eH), 3.28e3.21 (m, 1H, C7eH), 2.91e2.80 (m, 1H, C7eH), 2.74e2.58 (m, 2H, C8eH), 2.44 (s, 3H, N14eCH3).13C NMR (101 MHz, DMSO-d6): d 152.18, 140.54, 136.68, 131.40, 127.99, 126.19, 123.73, 121.04, 118.37, 117.93, 114.29, 112.45, 111.38, 110.10, 74.15 (C13b), 55.87 (C5), 50.03 (C7), 39.56 (N14eCH3), 20.87 (C8). HPLC purity ¼ 97.94%, tR ¼ 4.53 min. HRMS (ESI): calcd for [M þ H]þ (C19H20N3O) requires m/z 306.1601, found 306.1605. 4.4. General procedure for preparation of carbamates of 3hydroxyevodiamine (10aef) To the solution of 3-hydroxyevodiamine (8, 64 mg, 0.2 mmol) in dried THF (8 mL) were added diisopropylethylamine (52 mg, 68 mL, 0.4 mmol, 2.0 eq.) and the appropriate isocyanate (0.4 mmol, 2.0 eq.). The reaction mixture was stirred at r.t for 2e4 days; during stirring a solid precipitated. Then the mixture was concentrated in vacuo to remove about 3 mL of THF, the resulting white solid was collected by filtration and rinsed with diethyl ether to afford the target compound. 4.4.1. 14-Methyl-5-oxo-5,7,8,13,13b,14-hexahydroindolo[20 ,30 :3,4] pyrido[2,1-b]quinazolin-3-yl heptyl carbamate (10c) White solid (61 mg, 66%). 1H NMR (400 MHz, DMSO-d6) d 11.14 (s, 1H, N13eH), 7.72 (t, J ¼ 5.7 Hz, 1H, NHCOO), 7.49 (d, J ¼ 7.8 Hz, 1H), 7.46 (d, J ¼ 2.8 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.24 (dd, J ¼ 8.7, 2.8 Hz, 1H), 7.16e7.09 (m, 2H), 7.05e6.97 (m, 1H), 6.10 (s, 1H, C13bH), 4.68e4.59 (m, 1H, C7eH), 3.24e3.16 (m, 1H, C7eH), 3.04 (dd, J ¼ 13.0, 6.7 Hz, 2H, C10 H2), 2.94e2.78 (m, 2H, C8eH), 2.74 (s, 3H, N14eCH3), 1.53e1.40 (m, 2H, C20 H2), 1.27 (s, 8H, (CH2)4), 0.87 (t, J ¼ 6.8 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6): d 163.48 (C5), 154.43 (NHCOO), 146.30, 145.21, 136.59, 129.89, 127.09, 125.84, 121.89, 120.86, 120.36, 119.94, 118.87, 118.27, 111.63, 111.56, 69.38 (C13b), 40.44 (2 C, C7 and C10 ), 36.57 (N14eCH3), 31.19 (C20 ), 29.15 (C30 ), 28.34 (C40 ), 26.16 (C50 ), 22.01 (C60 ), 19.54 (C8), 13.92 (CH3). HPLC purity ¼ 100%, tR ¼ 9.62 min. HRMS (ESI): calcd for [M þ H]þ (C27H33N4O3) requires m/z 461.2547, found 461.2552. 4.5. General procedure for preparation of carbamates of 5-deoxo-3hydroxyevodiamine (11a-f) To the solution of 14-methyl-5,7,8,13,13b,14-hexahydroindolo [20 ,30 :3,4]pyrido[2,1-b]quinazolin-3-ol (61 mg, 0.2 mmol) in CH2Cl2 (15 mL) were added triethylamine (22 mg, 30.6 mL, 0.22 mmol, 1.1 eq.) and appropriate isocyanate (0.22 mmol, 1.1 eq.). The reaction mixture was stirred at r.t. for 20 h, and then the mixture was concentrated in vacuo to give viscous oils, which were purified by column chromatography (CH2Cl2/MeOH ¼ 10/1) to the products (usually as yellow foams). 4.5.1. 14-Methyl-5,7,8,13,13b,14-hexahydroindolo[20 ,30 :3,4]pyrido [2,1-b]quinazolin-3-yl heptyl carbamate (11c) Yellow foam (85 mg, yield 95%). 1H NMR (400 MHz, CDCl3) d 8.31 (s, 1H, N13eH), 7.54 (d, J ¼ 7.8 Hz, 1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.23e 7.17 (m, 1H), 7.13 (td, J ¼ 7.5, 1.0 Hz, 1H), 7.00 (d, J ¼ 8.7 Hz, 1H), 6.94
(dd, J ¼ 8.7, 2.5 Hz, 1H), 6.83 (d, J ¼ 2.5 Hz, 1H), 4.99 (t, J ¼ 5.7 Hz, 1H, heptyl-NH), 4.75 (s, 1H, C13b-H), 4.03 (d, J ¼ 15.2 Hz, 1H, C5eH), 3.82 (d, J ¼ 15.2 Hz, 1H, C5eH), 3.37e3.21 (m, 3H, C7eH & 2 C10 H2), 3.10e2.97 (m, 1H, C7eH), 2.85e2.71 (m, 2H, C8eH), 2.60 (s, 3H, N14eCH3), 1.62e1.52 (m, 2H, C20 H2), 1.41e1.27 (m, 8H, (CH2)4), 0.89 (t, J ¼ 6.4 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) d 155.06 (CO), 146.07, 145.92, 136.57, 130.86, 127.94, 126.98, 123.56, 122.27, 120.51, 119.87, 119.64, 118.68, 112.08, 111.25, 74.27 (C13b), 56.23 (C5), 50.71 (C7), 41.45 (C10 ), 39.78 (NeCH3), 31.88 (C20 ), 29.99 (C30 ), 29.08 (C40 ), 26.86 (C50 ), 22.73 (C60 ), 21.21 (C8), 14.21 (CH3). HPLC purity ¼ 99.19%, tR ¼ 7.88 min. HRMS (ESI): calcd for [M þ H] þ (C27H35N4O2) requires m/z 447.2755, found 447.2766. 4.6. Inhibition assay of AChE and BChE AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) and BChE (E.C.3.1.1.8, from equine serum) were purchased from Sigmae Aldrich (Steinheim, Germany). DTNB (Ellman’s reagent), ATC and BTC iodides were obtained from Fluka (Buchs, Switzerland). The assay was performed as described in the following procedure [18, 19, and 28]. Stock solutions of the test compounds were prepared in ethanol, 100 mL of which gave a final concentration of 103 M when diluted to the final volume of 1.66 mL. The highest concentration of the test compounds applied in the assay was 104 M (3% EtOH in the stock solution did not influence enzyme activity). In order to obtain an inhibition curve, at least five different concentrations (normally 104e109 M) of the test compound were measured at 25 C and 412 nm, each concentration in triplicate. For buffer preparation, 3.12 g of sodium dihydrogen phosphate (20 mmol) were dissolved in 500 mL of water and adjusted with NaOH to pH ¼ 8.0 0.1. Enzyme solutions were prepared to give 2.5 units mL1 in 1.4 mL aliquots. Furthermore, 0.01 M DTNB solution, 0.075 M ATC and BTC solutions, respectively, were used. A cuvette containing 1.5 mL of phosphate buffer, 50 mL of the respective enzyme, 10 mL of DTNB and 50 mL of the test compound solution was allowed to stand for 30 min, and the reaction was started by addition of 10 mL of the substrate solution (ATC/BTC). The solution was mixed immediately, and exactly 4.5 min after substrate addition the absorption was measured. For the reference value, 100 mL of water replaced the test compound solution. For determining the blank value, additionally 100 mL of water replaced the enzyme solution. The inhibition curve was obtained by plotting the percentage enzyme activity (100% for the reference) versus logarithm of test compound concentration. 4.7. ORAC assay The antioxidant activity was determined by the oxygen radical absorbance capacityefluorescein (ORAC-FL) assay [33], modified by Dávalos et al. [34]. The ORAC assay measures antioxidant scavenging activity against peroxyl radicals, their formation induced by 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH) at 37 C. The reaction was carried out in 75 mM phosphate buffer (pH 7.4) and the final reaction mixture was 200 mL. Antioxidant (20 mL) and fluorescein (120 mL, 300 nM final concentration) were placed in the wells of a 96 well plate and the mixture was incubated for 15 min at 37 C. Then AAPH (Sigma, Steinheim Germany) solution (60 mL; 12 mM final concentration) was added rapidly. The plate was immediately placed into a SpectraFluor Plus plate reader (Tecan, Crailsheim, Germany) and fluorescence measured every 60 s for 90 min with excitation at 485 nm and emission at 535 nm. 6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox, Sigma, Steinheim, Germany) was used as standard (1e8 mM, final
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
concentration). A blank (FL þ AAPH) using phosphate buffer instead of antioxidant and Trolox calibration were carried out in each assay. The samples were measured at different concentrations (1e5 mM). All reaction mixtures were prepared fourfold and at least four independent runs were performed for each sample. Fluorescence measurements were normalized to the curve of the blank (without antioxidant). From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated as:
AUC ¼ 1 þ
iX ¼ 90
fi =f0
(1)
i¼1
Where f0 is the initial fluorescence at 0 min and fi is the fluorescence at time i. The net AUC for a sample was calculated as follows:
Net AUC ¼ AUCantioxidant AUCblank
(2)
The ORAC-FL values were calculated:
½ðAUC Sample AUC blankÞ = ðAUC Trolox AUC blankÞ ½ðconcentration of Trolox=concentration of sampleÞ (3) and expressed as Trolox equivalents by using the standard curve calculated for each assay. Final results were in mM of Trolox equivalent/mM of compound. 4.8. Neuroprotection and neurotoxicity assay Cells and cell culture: HT-22 cells [29,30] were derived from murine hippocampal tissue [35] and were kindly provided by the Max Planck Institute of Psychiatry, Munich. HT-22 cells are grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (Biochrom, Berlin, Germany). Cells were kept under standard cell culture conditions at 37 C under 5% CO2 in a humidified incubator. Cells were subcultured every 2 days. Cell viability was determined in a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay [36] under recently described conditions [19,31,32]. Briefly, cells were seeded in 96well plates at a density of 5 103 per well and cultured for 24 h. Subsequently cells were incubated for another 24 h either with medium, compounds, or solvent only in presence (neuroprotection assay) or absence (neurotoxicity assay) of 5 mM glutamate (monosodium-L-glutamate, Merck, Darmstadt, Germany). Quercetin (Sigma, Steinheim, Germany) in a concentration of 25 mM served as positive control in the neuroprotection assay. MTT (Sigma, Steinheim, Germany) solution (4 mg/mL in PBS) was diluted 1:10 with medium and the mixture was added to the wells after removal of previous medium. The plates were then incubated for another 3 h. Afterward, supernatants were removed and 100 mL of lysis buffer (10% SDS) was added to the wells. Absorbance at 560 nM was determined on the next day with a multiwell plate photometer (SpectraFluor Plus, Crailsheim, Germany). Results of cell viability are expressed as percentage to untreated control cells. All compounds were dissolved in DMSO and diluted with fresh medium. DMSO concentration in final dilutions was 0.1%. Statistical Analysis: Data are expressed as means SD of at least 3 different independent experiments. Data were subjected to oneway ANOVA followed by Dunnett’s multiple comparison post test using GraphPad Prism 5 Software. (Levels of significance * p < 0.05; **p < 0.01; ***p < 0.001).
21
Acknowledgments M. Decker gratefully acknowledges the German Science Foundation (“Deutsche Forschungsgemeinschaft”) for financial support (DFG DE1546/6-1). Appreciation is expressed to Professor S. Elz at the University of Regensburg for providing his expertise and facilities for HRMS and kinetic studies. We thank Gabi Brunner for her excellent technical assistance on performing the ORAC assay. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.05.002. References [1] Y. Huang, L. Mucke, Cell 148 (2012) 1204e1222. [2] C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Lancet 377 (2011) 1019e1031. [3] A. Takeda, E. Loveman, A. Clegg, J. Kirby, J. Picot, E.C. Payne, Int. J. Geriatr. Psych. 21 (2006) 17e28. [4] M. Singh, M. Kaur, H. Kukreja, R. Chugh, O. Silakari, D. Singh, Eur. J. Med. Chem. 70 (2013) 165e168. [5] X. Chen, M. Decker, Curr. Med. Chem. 20 (2013) 1673e1685. [6] S. Darvesh, D.A. Hopkins, C. Geula, Nat. Rev. Neurosci. 4 (2003) 131e138. [7] E. Giacobini, Pharmacol. Res. 50 (2004) 433e440. [8] Y. Furukawa-Hibi, T. Alkam, A. Nitta, A. Matsuyama, H. Mizoguchi, K. Suzuki, S. Moussaoui, Q.S. Yu, N.H. Greig, T. Nagai, K. Yamada, Behav. Brain. Res. 225 (2011) 222e229. [9] N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu, J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi, D.K. Lahiri, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 17213e17218. [10] P.W. Elsinghorst, C.M. Tanarro, M. Gütschow, J. Med. Chem. 49 (2006) 7540e 7544. [11] S. Rizzo, C. Rivière, L. Piazzi, A. Bisi, S. Gobbi, M. Bartolini, V. Andrisano, F. Morroni, A. Tarozzi, J.P. Monti, A. Rampa, J. Med. Chem. 51 (2008) 2883e 2886. [12] C.G. Carolan, G.P. Dillon, D. Khan, S.A. Ryder, J.M. Gaynor, S. Reidy, J.F. Marquez, M. Jones, V. Holland, J.F. Gilmer, J. Med. Chem. 53 (2010) 1190e1199. [13] E. Rivera-Becerril, P. Joseph-Nathan, V.M. Pérez-Alvarez, M.S. Morales-Ríos, J. Med. Chem. 51 (2008) 5271e5284. [14] Q. Yu, H.W. Holloway, T. Utsuki, A. Brossi, N.H. Greig, J. Med. Chem. 42 (1999) 1855e1861. [15] D. Karlsson, A. Fallarero, G. Brunhofer, P. Guzik, M. Prinz, U. Holzgrabe, T. Erker, P. Vuorela, Eur. J. Pharm. Sci. 45 (2012) 169e183. [16] M. Decker, F. Krauth, J. Lehmann, Bioorg. Med. Chem. 14 (2006) 1966e1977. [17] X. Chen, I.G. Tikhonova, M. Decker, Bioorg. Med. Chem. 19 (2011) 1222e1235. [18] M. Decker, J. Med. Chem. 49 (2006) 5411e5413. [19] F.H. Darras, B. Kling, J. Heilmann, M. Decker, ACS Med. Chem. Lett. 3 (2012) 914e919. [20] A.M. Palmer, Trends Pharmacol. Sci. 32 (2011) 141e147. [21] S.H. Lee, J.K. Son, B. Se Jeong, T.C. Jeong, H.W. Chang, E.S. Lee, Y. Jahng, Molecules 13 (2008) 272e300. [22] L. Wang, C.P. Hu, P.Y. Deng, S.S. Shen, H.Q. Zhu, J.S. Ding, G.S. Tan, Y.J. Li, Planta Med. 71 (2005) 416e419. [23] G. Dong, S. Wang, Z. Miao, J. Yao, Y. Zhang, Z. Guo, W. Zhang, C. Sheng, J. Med. Chem. 55 (2012) 7593e7613. [24] J. Jiang, C. Hu, Molecules 14 (2009) 1852e1859. [25] W.F. Chiou, C.F. Chen, Eur. J. Pharmacol. 446 (2002) 151e159. [26] S.M. Yuan, K. Gao, D.M. Wang, X.Z. Quan, J.N. Liu, C.M. Ma, C. Qin, L.F. Zhang, Acta Pharm. Sin. 32 (2011) 295e302. [27] J.Y. Lee, M.R. Cha, C.W. Choi, Y.S. Kim, B.H. Lee, S.Y. Ryu, Korean J. Pharmacogn. 43 (2012) 122e126. [28] G.L. Ellman, K.D. Coutney, V. Anders, R.M. Featherstone, Biochem. Pharmacol. 7 (1961) 88e95. [29] J.B. Davis, P. Maher, Brain Res. 652 (1994) 169e173. [30] S. Tan, D. Schubert, P. Maher, Curr. Top. Med. Chem. 1 (2001) 497e506. [31] X. Chen, K. Zenger, A. Lupp, B. Kling, J. Heilmann, C. Fleck, B. Kraus, M. Decker, J. Med. Chem. 55 (2012) 5231e5242. [32] B. Kling, D. Bücher, P. Palatzky, F.M. Matysik, M. Decker, J. Wegener, J. Heilmann, J. Nat. Prod. 77 (2014) 446e454. [33] B. Ou, M. Hampsch-Woodill, R.J. Prior, J. Agric. Food. Chem. 49 (2001) 4619e 4926. [34] A. Dávalos, C. Gómez-Cordovés, B. Bartolomé, J. Agric. Food. Chem. 52 (2004) 48e54. [35] B.H. Morimoto, D.E. Koshland, Neuron 5 (1990) 875e880. [36] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Identification of a neuroprotective and selective butyrylcholinesterase inhibitor derived from the natural alkaloid evodiamine Guozheng Huang a, Beata Kling b, Fouad H. Darras b, Jörg Heilmann b, Michael Decker a, * a b
Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2013 Received in revised form 7 April 2014 Accepted 1 May 2014 Available online 4 May 2014
Two sets of carbamates based on the natural alkaloid evodiamine were designed, synthesized and evaluated as potential butyrylcholinesterase inhibitors. Although a set of carbamates of 3hydroxyevodiamine (10aef) is inactive both at AChE and BChE, carbamates of 5-deoxo-3hydroxyevodiamine (11aef) exhibit much better potency with selectivity toward BChE. The heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c) shows the best potency with an IC50 value of 77 nM and very good selectivity over AChE. ORAC and cell-based assays indicate 11c owns pronounced antioxidant properties with 1.75 Trolox equivalents and strong neuroprotection even from 1 mM onwards. These combined activities might enable compound 11c to be a potential candidate for treatment of Alzheimer’s disease. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Alzheimer’s disease Butyrylcholinesterase inhibitor Neuroprotection Evodiamine Carbamates
1. Introduction Alzheimer’s disease (AD), the most prominent form of dementia, is clinically defined as a progressive and irreversible loss of cognitive functions combined with neuropsychiatric disturbance. Although numerous efforts have been dedicated to the investigation of its etiologies, no effective method is yet available to prevent or reverse AD and its pathogenesis still remains unclear [1]. Several hypotheses have been advocated to constitute the key pathological hallmarks of AD, including b-amyloid deposits, s-protein aggregation, oxidative stress, and cholinergic system dysfunction [2]. According to the cholinergic hypothesis, drastic deficit of the neurotransmitter acetylcholine is the ultimate reason for failure in neurotransmission and subsequent aggravating cognitive and memory symptoms. Therefore, acetylcholinesterase (AChE) inhibitors are utilized clinically for the treatment of mild AD, such as tacrine, donepezil, rivastigmine and galantamine [3]. Current medicinal chemistry research focuses on novel AChE inhibitors, often with multifunctional and multi-target activities [4,5]. The second type of cholinesterase in the human body, butyrylcholinesterase (BChE), is also known as plasma cholinesterase. Although its portfolio of functions is still not completely
* Corresponding author. E-mail address: [email protected] (M. Decker). http://dx.doi.org/10.1016/j.ejmech.2014.05.002 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
understood, it may be also involved in the normal nervous system function and might participate in pathological processes in the etiology of AD as elucidated in several studies [6]. During later stage of AD, the activity of AChE decreases to reach 10e15% of normal values in certain brain regions, while the amount of BChE remains the same or even increases up to 2-fold [7]. Hence, selective AChE inhibitors might therefore encounter clinical ineffectiveness in later stages of AD. Actually, a balance between AChE and BChE inhibition or selectivity toward BChE might be more desirable. Furthermore, in vivo studies proved selective BChE inhibitors elevate brain acetylcholine, lower b-amyloid peptide, and ameliorate cognitive dysfunction [8,9]. In summary, selective BChE inhibitors could be of importance for treatment of AD and exploration of the pathogenesis of AD. Up to now, only a few selective BChE inhibitors (with selectivity index, SI > 100) were developed, such as heterobivalent tacrine derivatives [10], benzofurans [11] and isosorbide-based compounds [12] (the most potent one is isosorbide-2-benzyl carbamate-5salicylate, 1), cymserine analogs [13,14] (N1-phenylethyl-norcyserine, 2), and diarylimidazole compounds [15] (3). Our group used quinazolinimines as lead structures to develop a series of Nbridgeheaded tri- and tetracylic compounds with improved selectivity toward BChE [16e18]. Further exploration on the related structures identified a carbamate-based tetracyclic lead structure (4) [19]. Performing SARs on a library of tri- and tetracyclic structures with and without an additional carbamate moiety revealed
16
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
that a tetracyclic structure holds better potency and selectivity than the tricyclic structure. This result intrigued us to explore the possibility of better interaction between the aromatic ring and the binding region of BChE. It is well established that oxidative stress, the formation of reactive oxygen species (ROS), and subsequent neurotoxicity are key processes in the pathophysiology of AD [20]. We showed previously that compound 4 and its analogs possess very significant antioxidant properties (determined both with regard to their physico-chemical radical scavenging properties as well as with regard to neuron protection against oxidative stress) [19]. Evodiamine is the major quinazoline alkaloid isolated from Evodia rutaecarpa used in Traditional Chinese Medicine for treatment of a variety of ailments including headache, abdominal pain, postpartum hemorrhage, dysentery and amenorrhea [21,22]. Several research efforts indicate that evodiamine and its chemical derivatives are potent anti-cancer drug candidates [23,24]. The physiological effects of evodiamine on aged animals implicate a potential for the treatment of erectile dysfunction [25]. An in vivo study proved that it also can improve cognitive abilities in a transgenic mouse model of AD [26]. Recently, Lee et al. described a specific inhibitory activity on BChE with an IC50 value of 1.7 mg/mL (corresponding to 5.6 mM) although this value is far lower than for the compounds shown in Chart 1 [27]. In this study, we wanted to extend and continue our earlier studies seeking highly selective and potent BChE inhibitors with pronounced neuroprotective effects by combining the potent natural compound evodiamine with a privileged carbamate scaffold and applying the knowhow we had gained from previous tetracyclic scaffolds. 2. Results and discussion 2.1. Chemistry Condensation of 4,9-dihydro-3H-pyrido[3,4-b]indole (6) and 6(benzyloxy)-1-methyl-1H-benzo[d] [1,3]oxazine-2,4-dione (5) in refluxing dichloromethane afforded the desired 3benzyloxyevodiamine (7) in excellent yield (97%) (Scheme 1).
Fig. 2. Evaluation of neurotoxicity (A) and neuroprotection (B) of compounds 14, 9, 10c and 11c, respectively, against glutamate induced oxidative stress on HT-22 cells. Results are presented as means SD and refer to untreated control cells which were set as 100% values. Experiments were carried out with 4 parallels and repeated independently at least three times. Data were subjected to one-way ANOVA followed by Dunnet’s multiple comparison post-test using GraphPad Prism 5 Software. Levels of significance are: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
Intermediate 6 could be obtained from indole via a six-step linear synthesis. The isatoic anhydride derivative 5, which represents an activated form of anthranilic acid with a benzylated phenolic OH, was synthesized in five steps with almost quantitative overall yield starting from 5-hydroxyanthranilic acid (cf. Supplementary Material for the syntheses of the two intermediates). Benzylation of the phenolic group in compound 5 is necessary to avoid methylation of the phenolic group and to improve solubility in dichloromethane for subsequent fusion reaction. 3-Benzyloxyevodiamine (7) was
Fig. 1. (A) Time-dependent pattern of BChE inhibition by compound 11c (0.25e1 mM) and (B) Stability constants of the inhibitoreBChE complex (KC) and rate constants of carbamoyl-ChE formation (k3) of 11c and physostigmine, respectively.
Chart 1. Structures of selective BChE inhibitors.
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
17
Scheme 1. Synthetic protocol for target carbamates 10aef and 11aef. Reagents and conditions: (i) CH2Cl2, reflux, 20 h; (ii) 1 atm H2, THF, r.t., 20 h; (iii) LiAlH4, THF, r.t., 8 h; (iv) isocyanate, Hünig’s base, THF, 2e4 days; (v) isocyanate, triethylamine, CH2Cl2, 2e4 h.
debenzylated by hydrogenation to convert it into 3hydroxyevodiamine (8). Carbamoylation of 3-hydroxyevodiamine with appropriate isocyanates in the presence of Hünig’s base generated the target carbamates 10aef [19]. 3-Hydoxyevodiamine was reduced by lithium aluminum hydride to give 5-deoxo-3hydroxyevodiamine (9), which could also be carbamoylated using isocyanates in the presence of triethylamine to afford the second type of carbamates (11aef) (Scheme 1). Evodiamine (13) and 5deoxoevodiamine (14) were synthesized according to a recent literature method [23] (Scheme 2). The compounds’ structures and purities were fully supported by analytical and spectral data (1H NMR, 13C NMR, HPLC, and HRMS). 2.2. Pharmacology 2.2.1. Inhibition assay of AChE and BChE Synthesized carbamates and synthetic intermediates, together with evodiamine and 5-deoxoevodiamine, were investigated for inhibition of the hydrolase activity of AChE and BChE using Ellman’s reaction [19,28]. The results are summarized in Table 1. Our lead structure evodiamine (13) is moderately potent toward BChE with an IC50 of 4.62 mM and shows moderate selectivity, which is consistent with literature [27]. Reduction of evodiamine to 14 resulted in higher potency and better selectivity which indicates removal of 5-position’s amide oxygen is well tolerated for maintaining potency. In contrast to our expectation, introduction of 3hydroxy group into evodiamine rendered compound 8 to lose activity on both AChE and BChE. This is in strong contrast to the
respective benzo analog we previously described [19]. Nevertheless, reduction of 8 to 5-deoxo-3-hydroxyevodiamine 9 recovered potency, with moderate selectivity toward BChE. Surprisingly, carbamates of 3-hydroxyevodiamine (10aef) are not inhibiting either AChE or BChE at 10 mM, while after carbamoylation, the set of 5-deoxo-3-hydroxyevodiamine derivatives (11aef) are exhibiting better potency than 5-deoxo-3hydroxyevodiamine 9. Aromatic carbamates (11def) are less active with IC50 values higher than 10 mM (inhibition at higher concentrations could not be determined because of insolubility at >10 mM). Aliphatic carbamates (11aec) are more potent though, showing IC50 values in the micromolar and submicromolar range. The by far most potent and most selective BChE inhibitor is the heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c), which represents a 2-digit nanomolar inhibitor with no significant activity on AChE even at 10 mM. Its potency toward BChE was as good as the positive control physostigmine, but exhibiting pronounced BChE selectivity. 2.2.2. Kinetic study of BChE inhibition Rivastigmine (ExelonÒ) and the natural product physostigmine represent pseudo-irreversible inhibitors transferring the carbamate moiety to the serine unit in the active center of AChE and BChE (pseudo-irreversible, since the carbamate subsequently slowly hydrolyzes off the enzyme). Based on the structural similarity of 3-hydroxyevodiamine 11c and physostigmine, we assumed the 11c would own similar pseudo-irreversible inhibitory behavior. In order to obtain more detailed information about the time course
Scheme 2. Synthetic protocol for evodiamine (13) and 5-deoxoevodiamine (14) synthesis. Reagents and conditions: (i) CH2Cl2, reflux, 20 h; (ii) LiAlH4, THF, r.t., 8 h.
18
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
Table 1 AChE and BChE inhibition, selectivity index and antioxidant capacities expressed as Trolox equivalents (concentration range 1e5 mM). Test compound
IC50, mM (pIC50 SEM or inhibition in % at 10 mM) AChEa
Physostigmine 3-Benzyloxyevodiamine (7) 3-Hydroxyevodiamine (8) 5-Deoxo-3-hydroxyevodiamine (9) R ¼ ethyl (10a) R ¼ butyl (10b) R ¼ heptyl (10c)
(10d)
0.0077 3.01 >100 >100 >10 >10 >10
SIb
Trolox equ.
BChEa (8.11 0.08) (5.52 0.06) (29%) (43% at 100 mM) (15%) (10%) (13%)
0.060 0.880 >100 19.3 >10 >10 >10
(7.22 0.06) (6.07 0.05) (47% at 100 mM) (4.71 0.05) (21%) (12%) (1%)
>10 (10%)
>10 (15%)
>10 (14%)
>10 (7%)
>10 (15%)
>10 (3%)
0.13 3.42 >5.17
3.55 0.03
0.32 0.03
(10e)
(10f) R ¼ ethyl (11a) R ¼ butyl (11b) R ¼ heptyl (11c)
(11d)
>10 (26%) >10 (47%) >10 (17%)
1.03 (5.99 0.06) 3.97 (5.40 0.12) 0.077 (7.11 0.06
>9.71 >2.52 >>130
>10 (34%)
2.15 (5.67 0.10)
>4.65
>10 (13%)
>10 (41%)
>10 (19%)
>10 (30%)
>10 (37%) >10 (24%)
4.62 (5.34 0.10) 2.68 (5.57 0.09)
1.75 0.14
(11e)
(11f) Evodiamine (13) 5-Deoxoevodiamine (14) a b
>2.16 >3.73
2.32 0.24
AChE from electric eel and BChE from equine serum; data are the means of at least 3 independent determinations. Selectivity index [IC50(AChE)/IC50(BChE)].
of inhibition and therefore more precise data about the equilibrium constant of inhibition, a kinetic study of compound 11c (and physostigmine as positive control) was performed on BChE (Fig. 1) [19]. Compound 11c shows a time-dependent pattern of inhibition characterized by an increase until a steady state after 60 min. The equilibrium constant of the inhibitoreBChE complex (KC) and the carbamoylation rate constant (k3) of 11c were determined (Fig. 1). The heptyl carbamate 11c represents a highly potent pseudoirreversible BChE inhibitor with KC ¼ 492 nM. The rate constant of 0.243 min1 shows rapid carbamoylation.
carbamate transfer to the enzyme; therefore this potent antioxidant is the neuroprotectant to act in vivo. Surprisingly, 5deoxoevodiamine 14, without a free hydroxy group is also potent with 2.32 Trolox equivalents. Finally, carbamate 10c is almost inactive in the ORAC assay with only 0.32 Trolox equivalents (Table 1). All the indolo compounds exhibit significantly higher Trolox values compared to the previously described benzo compounds, this is especially true for the unsubstituted compound 5deoxoevodiamine 14, which shows the importance of the indole ring with regard to antioxidant properties [19].
2.2.3. Antioxidant capacities (ORAC assay) For evaluation of antioxidant potencies, in a first assay the target compounds’ radical scavenging capacities were determined by means of their ability to reduce the amount of peroxyl radicals (oxygen radical absorbance capacity assay, ORAC assay). The most potent compound 11c and its free hydroxy group containing starting material 9 were chosen. Also 5-deoxoevodiamine (14) was tested for comparison to screen the influence of the free hydroxy group. Compound 10c was tested to evaluate the influence of the 5position amide’s oxygen. As expected, 11c is potent with antioxidant capacity of 1.75 Trolox equivalents, meaning that it is more potent than the positive control Trolox, a water-soluble vitamin E derivative. 5-Deoxo-3-hydroxyevodiamine 9 with a free hydroxy group is much more potent with 3.55 Trolox equivalents, which indicates a free hydroxy group is favorable for antioxidant potency. The hydroxyl compound 9 is formed from compound 11c after
2.2.4. Neuroprotectivity To obtain physiologically relevant data of antioxidant compounds with regard to neuroprotection, the neuronal cell line HT22 was chosen as a model system, which is a glutamate sensitive cell line and derived from murine hippocampal tissue [29]. As these cells lack ionotropic glutamate receptors, addition of extracellular glutamate in high concentrations inhibits cystine (as oxidized form of cysteine) transport via the cystine/glutamate antiporter. This neuronal oxidative stress induced toxicity leads to intracellular cysteine and therefore glutathione depletion which induces intracellular ROS accumulation and cell injuries [30e32]. The same compounds selected for the ORAC assay (9, 10c, 11c, and 14) were subsequently evaluated at different concentrations ranging from 1 to 25 mM for their neuroprotective potential toward neuronal HT-22 cells exposed to glutamate and subsequent intracellular ROS generation and oxidative stress. Furthermore, the
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
compounds were tested for putative neurotoxic effects toward the HT-22 neurons to elucidate possible self-toxic effects toward the cells. Concerning potential neurotoxicity, neither compound 9 with its free hydroxy group nor the corresponding carbamate 11c significantly reduce the cells’ viability at concentrations 1, 5 or 10 mM but significantly reduce the viability of HT-22 neurons at 25 mM each (Fig. 2A). The same observation can be made for 5deoxoevodiamine 14. In contrast to these compounds, the carbamate 10c reveals significant neurotoxic effects by reducing the cells’ viability at all tested concentrations from 1 mM onward e this compound was inactive in the ORAC assay. As expected, compound 9 with the free hydroxy group that is also formed after BChE carbamoylation from compound 11c shows prominent neuroprotection toward the glutamate challenged HT22 cells nearly to the same extent as the very potent positive control quercetin from 5 mM onwards (Fig. 2B). The protective effect is slightly reduced with increasing concentrations, probably due to the neurotoxic effect which was measurable from 25 mM onwards (Fig. 2A). Again, also 5-deoxoevodiamine 14 showed neuroprotective effects at 10 and 25 mM. Therefore, the physico-chemical radical scavenging properties determined in the ORAC assay are reflected in the neuronal cell-based assay. It has been shown before that not only compounds with the free hydroxy group but also the corresponding carbamates can act as neuroprotectants in their own regards [19]. And indeed evaluation of the corresponding heptyl carbamate (11c) of compound 9 revealed strong neuroprotective properties even from 1 mM onwards to the same extent as compound 9 (Fig. 2B). Additionally, the heptyl carbamate 10c, which varies only in one oxygen atom from compound 11c, still owns slightly measurable neuroprotective properties, which might even have been more pronounced without the compounds self-toxicity toward the cells that was detected and is shown in Fig. 2A. In summary, with regard to biological activities, both ORAC and hippocampal neuronal cell-based assays indicate that the most potent BChE inhibitor 11c also acts as a potent neuroprotectant. It is worth to note that although the free hydroxy group is favorable for neuroprotectivity, an indole ring system containing molecules without free hydroxy group also remains active in its own regard.
19
High-resolution ESI mass (HRMS) spectral data were acquired on an Agilent 1100 series LC/MSD ion trap mass spectrometer and a Agilent 6520B UPLC-TOF-MS instrument (Agilent, Santa Clara, U.S.A.), respectively. Analytic HPLC was performed on a system from Shimadzu Products equipped with a DGU-20A3R controller, LC20AB liquid chromatograph, and a SPD-20A UV/Vis detector. Stationary phase was a Synergi 4U fusion-RP (150 4.6 mm) column. As mobile phase, a gradient MeOH-TFA (0.01%)/water-TFA (0.01%) (phase A/ phase B) were used. Gradient mode: 0e8 min (30e90% phase A), 8e 13 min (90% phase A), 15e18 min (90e30% phase A). Intermediates 5, 6, 12, evodiamine (13), and 5-deoxoevodiamine (14), respectively, were synthesized according to literature procedures describing individual steps, but not in the required sequence. Therefore, sequence and synthetic details can be found in the Supplementary Material. 4.1. Synthesis of 3-(benzyloxy)-14-methyl-7,8,13b,14tetrahydroindolo[20 ,30 :3,4] pyrido[2,1-b]quinazolin-5(13H)-one (3benzyloxyevodiamine, 7) A solution of 6-(benzyloxy)-1-methyl-1H-benzo[d][1,3] oxazine-2,4-dione (5, 1.416 g, 5 mmol) and 4,9-dihydro-3H-pyrido[3,4b]indole (6, 0.87 g, 5.11 mmol, 1.02 eq.) in CH2Cl2 (30 mL) was stirred under reflux for 20 h. Then it was concentrated, the residue was rinsed in diethyl ether to give the product as a yellow solid (1.93 g, 97%). 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, NH), 7.71 (d, J ¼ 2.7 Hz, 1H), 7.63e7.57 (m, 1H), 7.49e7.37 (m, 5H), 7.36e7.31 (m, 1H), 7.29e7.24 (m, 1H), 7.21e7.07 (m, 3H), 5.88 (t, J ¼ 1.4 Hz, 1H, C13b-H), 5.16e5.06 (m, 2H, PhCH2O), 4.91e4.84 (m, 1H, C7eH), 3.34e3.26 (m, 1H, C7eH), 3.03e2.88 (m, 2H, C8eH), 2.38 (s, 3H, NCH3). 13C NMR (101 MHz, CDCl3): d 164.60 (C5), 155.96, 144.72, 136.89, 136.87, 128.76 (2 C), 128.40, 128.21, 127.76 (2 C), 126.41, 125.18, 124.78, 123.19, 122.09, 120.17, 119.06, 113.68, 112.22, 111.48, 70.62 (PhCH2O), 69.22 (C13b), 39.55 (C7), 37.52 (NCH3), 20.31 (C8). HPLC purity ¼ 98.65%, tR ¼ 9.58 min. HRMS (ESI): calcd for [M þ H]þ (C26H24N3O2) requires m/z 410.1863, found 410.1867. 4.2. Synthesis of 3-hydroxy-14-methyl-7,8,13b,14-tetrahydroindolo [20 ,30 :3,4] pyrido[2,1-b]quinazolin-5(13H)-one (3hydroxyevodiamine, 8)
3. Conclusion By derivation of the biologically active natural alkaloid evodiamine two sets of carbamates were identified with BChE inhibitory properties. The heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c) showed the best potency with an IC50 value of 77 nM and excellent selectivity over BChE, representing one of the few compounds with such an activity and selectivity profile. Carbamate 11c and hydroxy compound 9 formed after carbamate transfer are very potent neuroprotectants. 4. Experimental Common reagents and solvents were obtained from commercial suppliers and used without further purification. Tetrahydrofuran (THF) was distilled from sodium/benzophenone under argon atmosphere. Reactions were conducted using dried flasks under an atmosphere of nitrogen. Reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254 plates (MachereyeNagel GmbH & Co. KG, Düren, Germany) and spots were detected under UV light (254 nm). Nuclear magnetic resonance spectra were recorded with a Bruker AV-400 NMR instrument (Bruker, Karlsruhe, Germany) in DMSO-d6 or CDCl3. Chemical shifts are expressed in ppm relative to CDCl3 or DMSO-d6 (7.26/2.50 and 77.16/39.52 ppm for 1H and 13C NMR, respectively).
3-Benzyloxyevodiamine (7, 1.85 g, 4.518 mmol) in dried THF (100 mL) was hydrogenated under 1 atm of hydrogen catalyzed by 500 mg palladium on carbon (10%) at r.t. for 20 h. The catalyst was filtered off, the filtrate was concentrated to afford the 3hydroxyevodiamine as a yellow solid (1.39 g, 97%). 1H NMR (400 MHz, DMSO-d6): d 11.24 (s, 1H, NH), 9.46 (s, 1H, 3-OH), 7.51 (d, J ¼ 7.8 Hz,1H), 7.37 (d, J ¼ 8.1 Hz,1H), 7.29 (d, J ¼ 2.9 Hz,1H), 7.15e7.09 (m, 1H), 7.07 (d, J ¼ 8.6 Hz, 1H), 7.05e7.00 (m, 1H), 6.96 (dd, J ¼ 8.6, 2.9 Hz, 1H), 5.95 (s, 1H, C13b-H), 4.70e4.56 (m, 1H, C7eH), 3.14e3.18 (m, 1H, C7eH), 2.93e2.86 (m, 1H), 2.85e2.75 (m, 1H, C8eH), 2.37 (s, 3H, N14eCH3). 13C NMR (101 MHz, DMSO-d6): d 163.66 (C5), 153.38, 142.58, 136.83, 129.36, 125.73, 123.75, 123.50, 121.87, 120.85, 118.81, 118.36, 113.08, 111.61, 111.57, 68.95 (C13b), 39.38 (C7), 36.95 (NCH3), 19.80 (C8). HPLC purity ¼ 99.71%, tR ¼ 7.73 min. HRMS (ESI): calcd for [M þ H]þ (C19H18N3O2) requires m/z 320.1394, found 320.1396. 4.3. Synthesis of 14-methyl-5,7,8,13,13b,14-hexahydroindolo [20 ,30 :3,4]pyrido[2,1-b] quinazolin-3-ol(3-hydroxy-5deoxoevodiamine, 9) To the cooled suspension of lithium aluminum hydride (0.61 g, 16 mmol, 4 eq.) in dried THF (40 mL) was added a solution of 3hydroxyevodiamine (8, 1.29 g, 4 mmol) in dried THF (40 mL) dropwise at 0 C. After addition, the mixture was stirred at r.t. for
20
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
8 h, and then cooled on an ice-bath again. To the cooled mixture was added NaSO4$10H2O in small portions until no gas was liberated any more. Then it was filtered through a pad of Celite, the cake washed with CH2Cl2/MeOH (5/1, 5 40 mL). The filtrate was combined and concentrated in vacuo to give a yellow oil which was purified by column chromatography (CH2Cl2/MeOH ¼ 10/1) to afford the product as a yellow solid (1.05 g, 86%). 1H NMR (400 MHz, DMSO-d6): d 11.06 (s, 1H, N13eH), 8.98 (s, 1H, 3-OH), 7.44 (d, J ¼ 7.8 Hz, 1H), 7.33 (d, J ¼ 8.0 Hz, 1H), 7.10e7.02 (m, 1H), 7.01e6.94 (m, 1H), 6.88 (d, J ¼ 8.7 Hz, 1H), 6.60 (dd, J ¼ 8.6, 2.8 Hz, 1H), 6.47 (d, J ¼ 2.7 Hz, 1H), 4.64 (s, 1H, C13b-H), 3.93 (d, J ¼ 15.3 Hz, 1H, C5eH), 3.69 (d, J ¼ 15.2 Hz, 1H, C5eH), 3.28e3.21 (m, 1H, C7eH), 2.91e2.80 (m, 1H, C7eH), 2.74e2.58 (m, 2H, C8eH), 2.44 (s, 3H, N14eCH3).13C NMR (101 MHz, DMSO-d6): d 152.18, 140.54, 136.68, 131.40, 127.99, 126.19, 123.73, 121.04, 118.37, 117.93, 114.29, 112.45, 111.38, 110.10, 74.15 (C13b), 55.87 (C5), 50.03 (C7), 39.56 (N14eCH3), 20.87 (C8). HPLC purity ¼ 97.94%, tR ¼ 4.53 min. HRMS (ESI): calcd for [M þ H]þ (C19H20N3O) requires m/z 306.1601, found 306.1605. 4.4. General procedure for preparation of carbamates of 3hydroxyevodiamine (10aef) To the solution of 3-hydroxyevodiamine (8, 64 mg, 0.2 mmol) in dried THF (8 mL) were added diisopropylethylamine (52 mg, 68 mL, 0.4 mmol, 2.0 eq.) and the appropriate isocyanate (0.4 mmol, 2.0 eq.). The reaction mixture was stirred at r.t for 2e4 days; during stirring a solid precipitated. Then the mixture was concentrated in vacuo to remove about 3 mL of THF, the resulting white solid was collected by filtration and rinsed with diethyl ether to afford the target compound. 4.4.1. 14-Methyl-5-oxo-5,7,8,13,13b,14-hexahydroindolo[20 ,30 :3,4] pyrido[2,1-b]quinazolin-3-yl heptyl carbamate (10c) White solid (61 mg, 66%). 1H NMR (400 MHz, DMSO-d6) d 11.14 (s, 1H, N13eH), 7.72 (t, J ¼ 5.7 Hz, 1H, NHCOO), 7.49 (d, J ¼ 7.8 Hz, 1H), 7.46 (d, J ¼ 2.8 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.24 (dd, J ¼ 8.7, 2.8 Hz, 1H), 7.16e7.09 (m, 2H), 7.05e6.97 (m, 1H), 6.10 (s, 1H, C13bH), 4.68e4.59 (m, 1H, C7eH), 3.24e3.16 (m, 1H, C7eH), 3.04 (dd, J ¼ 13.0, 6.7 Hz, 2H, C10 H2), 2.94e2.78 (m, 2H, C8eH), 2.74 (s, 3H, N14eCH3), 1.53e1.40 (m, 2H, C20 H2), 1.27 (s, 8H, (CH2)4), 0.87 (t, J ¼ 6.8 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6): d 163.48 (C5), 154.43 (NHCOO), 146.30, 145.21, 136.59, 129.89, 127.09, 125.84, 121.89, 120.86, 120.36, 119.94, 118.87, 118.27, 111.63, 111.56, 69.38 (C13b), 40.44 (2 C, C7 and C10 ), 36.57 (N14eCH3), 31.19 (C20 ), 29.15 (C30 ), 28.34 (C40 ), 26.16 (C50 ), 22.01 (C60 ), 19.54 (C8), 13.92 (CH3). HPLC purity ¼ 100%, tR ¼ 9.62 min. HRMS (ESI): calcd for [M þ H]þ (C27H33N4O3) requires m/z 461.2547, found 461.2552. 4.5. General procedure for preparation of carbamates of 5-deoxo-3hydroxyevodiamine (11a-f) To the solution of 14-methyl-5,7,8,13,13b,14-hexahydroindolo [20 ,30 :3,4]pyrido[2,1-b]quinazolin-3-ol (61 mg, 0.2 mmol) in CH2Cl2 (15 mL) were added triethylamine (22 mg, 30.6 mL, 0.22 mmol, 1.1 eq.) and appropriate isocyanate (0.22 mmol, 1.1 eq.). The reaction mixture was stirred at r.t. for 20 h, and then the mixture was concentrated in vacuo to give viscous oils, which were purified by column chromatography (CH2Cl2/MeOH ¼ 10/1) to the products (usually as yellow foams). 4.5.1. 14-Methyl-5,7,8,13,13b,14-hexahydroindolo[20 ,30 :3,4]pyrido [2,1-b]quinazolin-3-yl heptyl carbamate (11c) Yellow foam (85 mg, yield 95%). 1H NMR (400 MHz, CDCl3) d 8.31 (s, 1H, N13eH), 7.54 (d, J ¼ 7.8 Hz, 1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.23e 7.17 (m, 1H), 7.13 (td, J ¼ 7.5, 1.0 Hz, 1H), 7.00 (d, J ¼ 8.7 Hz, 1H), 6.94
(dd, J ¼ 8.7, 2.5 Hz, 1H), 6.83 (d, J ¼ 2.5 Hz, 1H), 4.99 (t, J ¼ 5.7 Hz, 1H, heptyl-NH), 4.75 (s, 1H, C13b-H), 4.03 (d, J ¼ 15.2 Hz, 1H, C5eH), 3.82 (d, J ¼ 15.2 Hz, 1H, C5eH), 3.37e3.21 (m, 3H, C7eH & 2 C10 H2), 3.10e2.97 (m, 1H, C7eH), 2.85e2.71 (m, 2H, C8eH), 2.60 (s, 3H, N14eCH3), 1.62e1.52 (m, 2H, C20 H2), 1.41e1.27 (m, 8H, (CH2)4), 0.89 (t, J ¼ 6.4 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) d 155.06 (CO), 146.07, 145.92, 136.57, 130.86, 127.94, 126.98, 123.56, 122.27, 120.51, 119.87, 119.64, 118.68, 112.08, 111.25, 74.27 (C13b), 56.23 (C5), 50.71 (C7), 41.45 (C10 ), 39.78 (NeCH3), 31.88 (C20 ), 29.99 (C30 ), 29.08 (C40 ), 26.86 (C50 ), 22.73 (C60 ), 21.21 (C8), 14.21 (CH3). HPLC purity ¼ 99.19%, tR ¼ 7.88 min. HRMS (ESI): calcd for [M þ H] þ (C27H35N4O2) requires m/z 447.2755, found 447.2766. 4.6. Inhibition assay of AChE and BChE AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) and BChE (E.C.3.1.1.8, from equine serum) were purchased from Sigmae Aldrich (Steinheim, Germany). DTNB (Ellman’s reagent), ATC and BTC iodides were obtained from Fluka (Buchs, Switzerland). The assay was performed as described in the following procedure [18, 19, and 28]. Stock solutions of the test compounds were prepared in ethanol, 100 mL of which gave a final concentration of 103 M when diluted to the final volume of 1.66 mL. The highest concentration of the test compounds applied in the assay was 104 M (3% EtOH in the stock solution did not influence enzyme activity). In order to obtain an inhibition curve, at least five different concentrations (normally 104e109 M) of the test compound were measured at 25 C and 412 nm, each concentration in triplicate. For buffer preparation, 3.12 g of sodium dihydrogen phosphate (20 mmol) were dissolved in 500 mL of water and adjusted with NaOH to pH ¼ 8.0 0.1. Enzyme solutions were prepared to give 2.5 units mL1 in 1.4 mL aliquots. Furthermore, 0.01 M DTNB solution, 0.075 M ATC and BTC solutions, respectively, were used. A cuvette containing 1.5 mL of phosphate buffer, 50 mL of the respective enzyme, 10 mL of DTNB and 50 mL of the test compound solution was allowed to stand for 30 min, and the reaction was started by addition of 10 mL of the substrate solution (ATC/BTC). The solution was mixed immediately, and exactly 4.5 min after substrate addition the absorption was measured. For the reference value, 100 mL of water replaced the test compound solution. For determining the blank value, additionally 100 mL of water replaced the enzyme solution. The inhibition curve was obtained by plotting the percentage enzyme activity (100% for the reference) versus logarithm of test compound concentration. 4.7. ORAC assay The antioxidant activity was determined by the oxygen radical absorbance capacityefluorescein (ORAC-FL) assay [33], modified by Dávalos et al. [34]. The ORAC assay measures antioxidant scavenging activity against peroxyl radicals, their formation induced by 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH) at 37 C. The reaction was carried out in 75 mM phosphate buffer (pH 7.4) and the final reaction mixture was 200 mL. Antioxidant (20 mL) and fluorescein (120 mL, 300 nM final concentration) were placed in the wells of a 96 well plate and the mixture was incubated for 15 min at 37 C. Then AAPH (Sigma, Steinheim Germany) solution (60 mL; 12 mM final concentration) was added rapidly. The plate was immediately placed into a SpectraFluor Plus plate reader (Tecan, Crailsheim, Germany) and fluorescence measured every 60 s for 90 min with excitation at 485 nm and emission at 535 nm. 6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox, Sigma, Steinheim, Germany) was used as standard (1e8 mM, final
G. Huang et al. / European Journal of Medicinal Chemistry 81 (2014) 15e21
concentration). A blank (FL þ AAPH) using phosphate buffer instead of antioxidant and Trolox calibration were carried out in each assay. The samples were measured at different concentrations (1e5 mM). All reaction mixtures were prepared fourfold and at least four independent runs were performed for each sample. Fluorescence measurements were normalized to the curve of the blank (without antioxidant). From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated as:
AUC ¼ 1 þ
iX ¼ 90
fi =f0
(1)
i¼1
Where f0 is the initial fluorescence at 0 min and fi is the fluorescence at time i. The net AUC for a sample was calculated as follows:
Net AUC ¼ AUCantioxidant AUCblank
(2)
The ORAC-FL values were calculated:
½ðAUC Sample AUC blankÞ = ðAUC Trolox AUC blankÞ ½ðconcentration of Trolox=concentration of sampleÞ (3) and expressed as Trolox equivalents by using the standard curve calculated for each assay. Final results were in mM of Trolox equivalent/mM of compound. 4.8. Neuroprotection and neurotoxicity assay Cells and cell culture: HT-22 cells [29,30] were derived from murine hippocampal tissue [35] and were kindly provided by the Max Planck Institute of Psychiatry, Munich. HT-22 cells are grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (Biochrom, Berlin, Germany). Cells were kept under standard cell culture conditions at 37 C under 5% CO2 in a humidified incubator. Cells were subcultured every 2 days. Cell viability was determined in a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay [36] under recently described conditions [19,31,32]. Briefly, cells were seeded in 96well plates at a density of 5 103 per well and cultured for 24 h. Subsequently cells were incubated for another 24 h either with medium, compounds, or solvent only in presence (neuroprotection assay) or absence (neurotoxicity assay) of 5 mM glutamate (monosodium-L-glutamate, Merck, Darmstadt, Germany). Quercetin (Sigma, Steinheim, Germany) in a concentration of 25 mM served as positive control in the neuroprotection assay. MTT (Sigma, Steinheim, Germany) solution (4 mg/mL in PBS) was diluted 1:10 with medium and the mixture was added to the wells after removal of previous medium. The plates were then incubated for another 3 h. Afterward, supernatants were removed and 100 mL of lysis buffer (10% SDS) was added to the wells. Absorbance at 560 nM was determined on the next day with a multiwell plate photometer (SpectraFluor Plus, Crailsheim, Germany). Results of cell viability are expressed as percentage to untreated control cells. All compounds were dissolved in DMSO and diluted with fresh medium. DMSO concentration in final dilutions was 0.1%. Statistical Analysis: Data are expressed as means SD of at least 3 different independent experiments. Data were subjected to oneway ANOVA followed by Dunnett’s multiple comparison post test using GraphPad Prism 5 Software. (Levels of significance * p < 0.05; **p < 0.01; ***p < 0.001).
21
Acknowledgments M. Decker gratefully acknowledges the German Science Foundation (“Deutsche Forschungsgemeinschaft”) for financial support (DFG DE1546/6-1). Appreciation is expressed to Professor S. Elz at the University of Regensburg for providing his expertise and facilities for HRMS and kinetic studies. We thank Gabi Brunner for her excellent technical assistance on performing the ORAC assay. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.05.002. References [1] Y. Huang, L. Mucke, Cell 148 (2012) 1204e1222. [2] C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Lancet 377 (2011) 1019e1031. [3] A. Takeda, E. Loveman, A. Clegg, J. Kirby, J. Picot, E.C. Payne, Int. J. Geriatr. Psych. 21 (2006) 17e28. [4] M. Singh, M. Kaur, H. Kukreja, R. Chugh, O. Silakari, D. Singh, Eur. J. Med. Chem. 70 (2013) 165e168. [5] X. Chen, M. Decker, Curr. Med. Chem. 20 (2013) 1673e1685. [6] S. Darvesh, D.A. Hopkins, C. Geula, Nat. Rev. Neurosci. 4 (2003) 131e138. [7] E. Giacobini, Pharmacol. Res. 50 (2004) 433e440. [8] Y. Furukawa-Hibi, T. Alkam, A. Nitta, A. Matsuyama, H. Mizoguchi, K. Suzuki, S. Moussaoui, Q.S. Yu, N.H. Greig, T. Nagai, K. Yamada, Behav. Brain. Res. 225 (2011) 222e229. [9] N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu, J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi, D.K. Lahiri, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 17213e17218. [10] P.W. Elsinghorst, C.M. Tanarro, M. Gütschow, J. Med. Chem. 49 (2006) 7540e 7544. [11] S. Rizzo, C. Rivière, L. Piazzi, A. Bisi, S. Gobbi, M. Bartolini, V. Andrisano, F. Morroni, A. Tarozzi, J.P. Monti, A. Rampa, J. Med. Chem. 51 (2008) 2883e 2886. [12] C.G. Carolan, G.P. Dillon, D. Khan, S.A. Ryder, J.M. Gaynor, S. Reidy, J.F. Marquez, M. Jones, V. Holland, J.F. Gilmer, J. Med. Chem. 53 (2010) 1190e1199. [13] E. Rivera-Becerril, P. Joseph-Nathan, V.M. Pérez-Alvarez, M.S. Morales-Ríos, J. Med. Chem. 51 (2008) 5271e5284. [14] Q. Yu, H.W. Holloway, T. Utsuki, A. Brossi, N.H. Greig, J. Med. Chem. 42 (1999) 1855e1861. [15] D. Karlsson, A. Fallarero, G. Brunhofer, P. Guzik, M. Prinz, U. Holzgrabe, T. Erker, P. Vuorela, Eur. J. Pharm. Sci. 45 (2012) 169e183. [16] M. Decker, F. Krauth, J. Lehmann, Bioorg. Med. Chem. 14 (2006) 1966e1977. [17] X. Chen, I.G. Tikhonova, M. Decker, Bioorg. Med. Chem. 19 (2011) 1222e1235. [18] M. Decker, J. Med. Chem. 49 (2006) 5411e5413. [19] F.H. Darras, B. Kling, J. Heilmann, M. Decker, ACS Med. Chem. Lett. 3 (2012) 914e919. [20] A.M. Palmer, Trends Pharmacol. Sci. 32 (2011) 141e147. [21] S.H. Lee, J.K. Son, B. Se Jeong, T.C. Jeong, H.W. Chang, E.S. Lee, Y. Jahng, Molecules 13 (2008) 272e300. [22] L. Wang, C.P. Hu, P.Y. Deng, S.S. Shen, H.Q. Zhu, J.S. Ding, G.S. Tan, Y.J. Li, Planta Med. 71 (2005) 416e419. [23] G. Dong, S. Wang, Z. Miao, J. Yao, Y. Zhang, Z. Guo, W. Zhang, C. Sheng, J. Med. Chem. 55 (2012) 7593e7613. [24] J. Jiang, C. Hu, Molecules 14 (2009) 1852e1859. [25] W.F. Chiou, C.F. Chen, Eur. J. Pharmacol. 446 (2002) 151e159. [26] S.M. Yuan, K. Gao, D.M. Wang, X.Z. Quan, J.N. Liu, C.M. Ma, C. Qin, L.F. Zhang, Acta Pharm. Sin. 32 (2011) 295e302. [27] J.Y. Lee, M.R. Cha, C.W. Choi, Y.S. Kim, B.H. Lee, S.Y. Ryu, Korean J. Pharmacogn. 43 (2012) 122e126. [28] G.L. Ellman, K.D. Coutney, V. Anders, R.M. Featherstone, Biochem. Pharmacol. 7 (1961) 88e95. [29] J.B. Davis, P. Maher, Brain Res. 652 (1994) 169e173. [30] S. Tan, D. Schubert, P. Maher, Curr. Top. Med. Chem. 1 (2001) 497e506. [31] X. Chen, K. Zenger, A. Lupp, B. Kling, J. Heilmann, C. Fleck, B. Kraus, M. Decker, J. Med. Chem. 55 (2012) 5231e5242. [32] B. Kling, D. Bücher, P. Palatzky, F.M. Matysik, M. Decker, J. Wegener, J. Heilmann, J. Nat. Prod. 77 (2014) 446e454. [33] B. Ou, M. Hampsch-Woodill, R.J. Prior, J. Agric. Food. Chem. 49 (2001) 4619e 4926. [34] A. Dávalos, C. Gómez-Cordovés, B. Bartolomé, J. Agric. Food. Chem. 52 (2004) 48e54. [35] B.H. Morimoto, D.E. Koshland, Neuron 5 (1990) 875e880. [36] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
