Bioactivity-guided synthesis of tropine derivatives as new agonists for melatonin receptors

Xiu-Juan Yinab, Chang-An Genga, Xiao-Yan Huanga, Hao Chena, Yun-Bao Maa, Xing-Long Chenab, Chang-Li Sunab, Tong-Hua Yangab, Jun Zhoua, Xue-Mei Zhanga and Ji-Jun Chen*a
aState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China. E-mail: chenjj@mail.kib.ac.cn; Fax: +86-871-6522-7197; Tel: +86-871-6522-3265
bUniversity of Chinese Academy of Sciences, Beijing 100049, PR China

Received 14th March 2016 , Accepted 18th April 2016

First published on 20th April 2016


Abstract

Twenty-three tropine derivatives as new melatonin receptor (MT1 and MT2) agonists were synthesized and evaluated on HEK293 cells in vitro. Derivatives 1f, 1i, 1j, 1m–1s and 1t exhibited increased agonisting activities on MT1 and MT2 receptors compared to the substrate tropine. Particularly, compound 1r showed significant agonistic activities on MT1 and MT2 receptors with EC50 values of 0.20 and 0.24 mM, respectively. The preliminary structure–activity relationships (SARs) of tropine derivatives were summarized for further investigation on melatonin receptor agonists.


Melatonin (MT), 5-methoxy-N-acetyltryptamine, released from the pineal gland at night, can regulate physiology and behavior by targeting MT1 and MT2 receptors.1 MT1 and MT2 receptors belonging to the G-protein-coupled receptor (GPCR) superfamily are widely distributed in brain and peripheral nervous systems of mammals,2–7 which are fascinating targets for drug discovery due to their multiply regulatory functions.8–10 MT1 receptor, generally expressed in the central nervous system and many peripheral tissues,11 modulates neuronal firing. MT2 receptor is observed in brain and lung, closely related to circadian rhythms of neuronal firing in the suprachiasmatic nucleus and dopamine release in retina.12 In recent years, different types of MT1 and MT2 receptors agonistic ligands were reported, some of which had been used for psychiatric purposes.13,14 For example, TAK-375 and VEC-162 were marketed for the treatment of insomnia,15 and circadian rhythm sleep disorders.16 However, most of the MT1 and MT2 receptors agonists are synthetic compounds possessing high similarity with melatonin,17 and exploring novel types of agonists is still needed.

In the 19th and early 20th centuries, tropane alkaloids attracted particular interest due to their potent and extensive biological activities,18 such as regulating secretion of the monoamine neurotransmitter,19–22 influencing expression of the glycine receptor,23 and modulating activation of the acetylcholine receptor.24–26 Tropine as a natural tropane alkaloid mainly distributed in Solanaceae plants, showed pleiotropic physiological effects in humans and animals,27 however, its agonistic effects on MT receptors have not been revealed.

Within the G-protein coupled receptor family of proteins, the MT1 and MT2 receptors can couple to multiple and distinct signal transduction cascades whose activation lead to unique cellular response.28 MT1 (or MT2)-expressing cell line was made in the HEK 293-Gα15 host cells, which supported high levels of recombinant MT1 (or MT2) expression on the cell surface and contained high levels of the promiscuous G protein Gα15 to enhance coupling of the receptor to the calcium signaling pathway. Based on the Ca2+ influx, the receptor channel pore could be controlled by agonists using fluorimetric techniques, monitored by Ca2+-sensitive dyes.29 Test data for detecting changes in intracellular calcium was the transient calcium flux observed after activation of G-coupled protein receptors.30 The details of Fluo-8 calcium assay were listed.31 Our previous bio-assay evaluation suggested that tropine exhibited moderate agonistic activities on MT1 and MT2 receptors with the values of 90.50% and 77.48% at 1 mM. Thus, a series of tropine derivatives were synthesized for developing new MT receptors agonists. The preliminary structure–activity relationships (SARs) were also discussed based on the bioassay on HEK293 cells in vitro.

With an objective to obtain target compounds which have agonistic activities on HEK293 cells in vitro, the synthetic strategies for target compounds are flexible. Tropine was reacted with various organic acids by Steglich esterification in the mixed solution of a catalytic amount of 4-dimethylaminopyridine (DMAP), dehydrating agent N,N-dicyclohexylcarbodiimide (DCC) and anhydrous CH2Cl2 to generate compounds 1a–f and 1i–l (Scheme 1).


image file: c6ra06748d-s1.tif
Scheme 1 Reagents and conditions: (a) DMAP, DCC, CH2Cl2, r.t., 60–70% for compounds 1a–x. (b) PCC, CH2Cl2, r.t., 95% for compound 2. (c) CH3CN, reflux, 50–65% for compounds 1m.

The results of preliminary bioassay on MT1 receptor showed that introduction of a benzoyl group and a cinnamoyloxy group improved agonistic activity compared with that of tropine (Fig. 1), and thus, derivatives 1g–h and 1n–x were gained for further study. Derivative 2 was synthesized starting from tropine with the oxidizing agent pyridinium chlorochromate (PCC) by oxidation reaction, which was no activity on MT1 receptor. Derivative 1c and piperazine were dissolved in CH3CN with reflux condition to give derivative 1m, and agonistic activity of derivative 1m on MT1 receptor increased. The structure of target compounds were identified by 1H-NMR, 13C-NMR, MS and HRMS. Physicochemical properties (including clog p – calculated logarithm of partition coefficient between n-octanol and water, TPSA-polar surface area, log S – a unit stripped logarithm of the solubility measured in mol liter−1) and toxicity profiles (including mutagenic effect, tumorigenic effect, irritating effect and reproductive effect) of these compounds were calculated and predicted using OSIRIS Property Explorer software at URL http://www.organic-chemistry.org/prog/peo/.32,33 The calculated data were shown in Table 1.


image file: c6ra06748d-f1.tif
Fig. 1 Agonistic activities of derivatives 2, 1a–f and 1i–m on MT1 receptor. The agonistic activities of derivatives (1.00 mM) and agomelatine (3.33 μM, the control group) were performed in triplicate, and the results were presented as [X with combining macron] ± SD (n = 3). The agonistic activity of the control group was 100%.
Table 1 Calculated physicochemical properties and predicted toxicity of compounds 2 and 1a–x
Compd clog[thin space (1/6-em)]pa TPSAb2) log[thin space (1/6-em)]Sc Toxicity risksd
M/T/I/R
a clog[thin space (1/6-em)]p, calculated logarithm of partition coefficient between n-octanol and water.b TPSA, polar surface area.c S, solubility.d Toxicity risks: M, mutagenic effect; T, tumorigenic effect; I, irritating effect; R, reproductive effect; L, low; M, medium; H, high.
2 0.65 20.31 −1.26 L/L/L/L
1a 0.99 29.54 −1.62 L/L/L/L
1b 1.89 29.54 −2.16 L/L/L/L
1c 2.12 29.54 −2.56 M/H/L/H
1d 2.57 29.54 −2.92 L/L/H/L
1e 6.44 29.54 −4.86 L/L/L/L
1f 2.43 29.54 −2.79 L/L/L/L
1g 3.04 29.54 −3.52 L/L/L/L
1h 3.16 29.54 −3.62 L/L/L/L
1i 2.25 29.54 −2.05 L/L/H/L
1j 2.76 29.54 −3.16 L/L/H/L
1k 2.46 38.77 −2.84 L/L/H/L
1l 1.42 67.87 −2.49 L/L/L/L
1m 2.32 32.78 −2.24 L/L/L/L
1n 3.10 29.54 −3.50 L/L/L/L
10 3.10 29.54 −3.50 L/L/L/L
1p 2.86 29.54 −3.47 L/L/L/M
1q 2.86 29.54 −3.47 L/L/L/L
1r 2.86 29.54 −3.47 L/L/L/L
1s 3.37 29.54 −3.89 L/L/L/L
1t 2.69 38.77 −3.17 L/L/H/M
1w 3.97 29.54 −4.63 L/L/L/L
1x 2.62 48.00 −3.19 L/L/L/L


As shown in Table 2, tropine displayed moderate agonistic activities on MT1 and MT2 receptors with the values of 90.50% and 77.48% at 1 mM. When the hydroxyl group was changed to be a carbonyl group, derivative 2 obviously reduced activities on MT1 and MT2 receptors with the values of 18.42% and 26.73% at 1 mM respectively, suggesting C[double bond, length as m-dash]O group was unfavorable activity for maintaining activities (Fig. 2). In an effort to gain more information of the SARs of tropine derivatives, the esterified products were further obtained.

Table 2 Agonistic activities of tropine derivatives (general molecular formula 1, Fig. 3) on melatonin receptorsa
Comp. R Agonistic activities (%)
MT1b MT2c
a Agomelatine was tested at the concentration of 3.33 μM and other compounds were tested at the concentration of 1.00 mM.b The agonistic activities expressed as [X with combining macron] ± SD (n = 3) were obtained by comparing to the highest agonistic activity that was achieved for the agomelatine (3.33 μM) and was set as 100%.c
Ago. 100.00 ± 3.54 100.00 ± 2.00
2 18.42 ± 5.72 26.73 ± 1.84
Tropine H 90.50 ± 3.33 77.48 ± 5.67
1a Acetyl 69.51 ± 3.43 98.45 ± 4.35
1b Butyryl 21.63 ± 3.70 149.97 ± 33.86
1c 4-Chloro butyryl −3.06 ± 0.45 106.34 ± 8.71
1d 2-Methylvaleryl 73.52 ± 4.62 141.05 ± 13.91
1e Myristoyl 4.20 ± 1.26 88.60 ± 14.28
1f Benzoyl 194.22 ± 3.15 219.61 ± 7.28
1g 4-Chlorlbenzoyl 100.44 ± 3.50 127.05 ± 15.81
1h 4-Bromoxybenzoyl 47.65 ± 1.74 88.69 ± 2.03
1i Senecioyl 163.13 ± 5.70 173.01 ± 50.85
1j Cinnamoyl 197.18 ± 14.55 215.67 ± 14.01
1k 3-Phenoxy-propionyl −2.55 ± 0.24 193.87 ± 7.35
1l Boc-glycyl 96.37 ± 2.97 95.64 ± 1.91
1m 4-(N-Piperidyl)butyryl 173.02 ± 2.35 217.84 ± 12.70



image file: c6ra06748d-f2.tif
Fig. 2 Structure–activity relationships of tropane derivatives.

image file: c6ra06748d-f3.tif
Fig. 3 General molecular formula 1 and 2.

Most of esterified derivatives showed attractively agonistic activities on MT1 and MT2 receptors (Table 2). The acetylated derivative 1a displayed similar agonistic activities compared with tropine. Derivative 1b, with a butyryl group at C-3 position, exhibited increased agonistic activity on MT2 receptor. Derivative 1c with a chlorinated group at the C-4 position of the butyryl group showed similar agonistic activity with derivative 1b. Derivative 1d with a 2-methyl-valeryl group owned an approximately 2-fold agonistic activity on MT2 receptor and similar agonistic activity on MT1 receptor compared to tropine. However, the myristoyl derivative 1e resulted in a significantly decreased activities on MT1 and MT2 receptors. The above analyses suggested that bearing –OCO– ester group at C-3 position and a side chain (less than 5 carbons) are crutial for agonistic activities (Fig. 2). Followingly, the effect of some bulky aromatic groups at C-3 position was investigated. As shown in Table 2, introduction of a benzoyl group improved agonistic activities on both receptors. Derivative 1f endowed significantly agonistic activities on MT1 (194.22% at 1.00 mM) and MT2 (219.61%, at 1.00 mM) receptors. These results encouraged us to further explore derivatives 1g and 1h by introducing diverse substitutions on a phenyl group. Interestingly, the unsubstituted benzoyl derivative 1f, showed higher agonistic activities than derivatives 1g and 1h with para-substituted at the phenyl ring. Agonistic activities of derivative 1i with a conjugate double bond was two times higher than that of the tropine on both receptors. Cinnamoyloxy substituted derivatives 1j with a conjugate double bond exhibited excellent agonistic activities on both receptors (Fig. 2), which encouraged us to further explore derivatives 1o–1x. Derivative 1k with a phenoxylpropionyl group showed a 3-fold agonistic activity on MT2 receptor but no activity on MT1 receptor. Derivative 1i with a N-(tert-butoxycarbonyl)-glycinyl group had similar agonistic activities compared with that of tropine. Derivative 1m with a 4-butyryl piperidyl group at C-4 position, displayed an approximately 2-fold agonistic activity on MT1 receptor and 3-fold on MT2 receptor compared with tropine.

Derivative 1j with a cinnamoyloxy group at C-3 position exhibited excellent agonistic activities on MT1 (197.18%, at 1.00 mM) and MT2 (215.67%, at 1.00 mM) receptors, suggesting that the cinnamoyl group is preferable for gaining high agonistic activity. Followingly, derivatives with diverse substitutions on the cinnamoyl group were synthesized in the following investigation. Derivatives 1n–x, with different substituents on the cinnamoyl ring, displayed agonistic activities from 56.65 to 190.04% on MT1, and from 30.53% to 255.23% on MT2, respectively (Table 3). Agonistic activities of methyl substituted analogues at ortho- and para-positions of phenyl ring (1n, 1o) resulted in a significantly decreased activity on MT1 receptor and maintained almost no change on MT2 receptor compared with derivative 1j. Fluorinated analogous (1p, 1q, 1r) with ortho-, meta-, and para-substituted patterns at the phenyl ring, displayed agonistic potency on MT2 receptor with the values of 177.57%, 196.72% and 255.23%, respectively. Agonistic activity of fluorinated analogue at para-position of phenyl ring exhibited higher activity than derivative 1j on MT2 receptor. Chlorinated derivative (1s) at meta-position of phenyl ring displayed slightly lower activity than derivative 1j on both receptors. Derivative 1t with a methoxy group at the C-3 position of the phenyl ring showed similar agonistic activities on both receptors compared with derivative 1j. The dichloro substituted analogue (1w) and the dimethoxy substituted analogue (1x) resulted in remarkably reduced activity on both receptors compared with derivative 1j.

Table 3 Agonistic activities of cinnamoyloxytropane derivatives (general molecular formula 2, Fig. 3) on melatonin receptorsa
Comp. R1 R2 R3 Agonistic activities (%)
MT1b MT2c
a Agomelatine was tested at the concentration of 3.33 μM and other compounds were tested at the concentration of 1.00 mM.b The agonistic activities expressed as [X with combining macron] ± SD (n = 3) were obtained by comparing to the highest agonistic activity that was achieved for the agomelatine (3.33 μM) and was set as 100%.c
Ago. 100.00 ± 3.54 100.00 ± 2.00
1n Me H H 158.60 ± 9.63 200.66 ± 4.50
1o H H Me 145.56 ± 2.76 223.44 ± 10.94
1p F H H 142.46 ± 6.25 177.57 ± 12.79
1q H F H 160.17 ± 9.02 196.72 ± 3.75
1r H H F 190.04 ± 9.13 255.23 ± 10.01
1s H Cl H 125.35 ± 2.64 183.36 ± 2.35
1t H H OMe 146.35 ± 10.32 193.24 ± 8.02
1w Cl H Cl 56.65 ± 8.75 36.56 ± 10.02
1x H OMe OMe 63.00 ± 6.26 30.53 ± 2.81


The dose–response curves for the most potent derivative 1r was investigated to provide EC50 values of 0.20 and 0.24 mM on MT1 and MT2 receptors, respectively (Fig. 4). Dose–response of calcium activity was performed in triplicate and monitored with FlexStation plate reader. EC50 values for the derivative 1r were determined from the dose–response curves obtained with eight concentrations from the range of 7.81 to 1000 μM for MT1 and MT2 receptors, and calculated by the software of Graphpad Prism 5.0.


image file: c6ra06748d-f4.tif
Fig. 4 The dose-dependent effects of derivative 1r on MT1 and MT2 receptors.

Conclusions

In conclusion, twenty-three derivatives of tropine were synthesized and evaluated on MT1 and MT2 receptors, of which, derivatives 1f, 1j and 1r exhibited promising agonistic activities. The preliminary SARs suggested that the hydroxyl group at C-3 position was crucial for maintaining agonistic activities, and esterification on the hydroxyl group will generate different activities. This investigation will provide valuable insights for further developing tropine derivatives as potential melatonin receptors agonists.

Author contributions

Xiu-juan Yin and Ji-jun Chen contributed to the design of experiments and the manuscript preparation. Xiu-juan Yin, Chang-an Geng, Xiao-yan Huang, Yun-bao Ma and Ji-jun Chen performed the experimental studies and analyzed the data. Xiao-yan Huang and Yun-bao Ma contributed to the design and test of biological activity. Xing-long Chen was responsible for data testing of HRMS. Chang-li Sun, Tong-hua Yang, Jun Zhou and Xue-mei Zhang contributed in critical reading and discussion on the manuscript. All the authors approved the final version.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81573322), the Program of Yunling Scholar, the Hundred Talents Program of the Chinese Academy of Sciences (CAS) and the Youth Innovation Promotion Association, CAS.

Notes and references

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06748d

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