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

Article abstract of DOI:10.1039/c6ra06748d

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.

Full text of DOI:10.1039/c6ra06748d

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Bioactivity-guided synthesis of tropine derivatives as new agonists
for melatonin receptors
Received 00th January 20xx,
Accepted 00th January 20xx
Xiu-Juan Yina, b, Chang-An Genga , Xiao-Yan Huanga , Hao Chena , Yun-Bao Maa , Xing-Long Chena, b
,
Chang-Li Suna,b , Tong-Hua Yanga,b , Jun Zhoua , Xue-Mei Zhanga and Ji-Jun Chena,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Twenty-three tropine derivatives as new melatonin receptors particular interest due to their potent and extensive biological
(MT1 and MT2) agonists were synthesized and evaluated on HEK293 activities,18 such as regulating secretion of the monoamine
19-22
cells in vitro. Derivatives 1f, 1i, 1j, 1m-1s and 1t exhibited increased neurotransmitter,
influencing expression of the glycine
agonisting activities on MT1 and MT2 receptors compared to the receptor,23 and modulating activation of the acetylcholine
substrate tropine. Particularly, compound 1r showed significantly receptor.24-26 Tropine as
natural tropane alkaloid mainly
a
agonistic activities on MT1 and MT2 receptors with EC50 values of distributed in Solanaceae plants, showed pleiotropic physiological
0.20 and 0.24 mΜ, respectively. The preliminary structure-activity effects in humans and animals,27 however, its agonistic effects on
relationships (SARs) of tropine derivatives were summarized for MT receptors have not been revealed.
further investigation on melatonin receptor agonists.
Within the G-protein coupled receptor family of proteins, the MT1
Melatonin (MT), 5-methoxy-N-acetyltryptamine, released from and MT2 receptors can couple to multiple and distinct signal
the pineal gland at night, can regulate a variety of physiology and transduction cascades whose activation lead to unique cellular
28
behavior by targeting MT1 and MT2 receptors.1 MT1 and MT2 response. MT1 (or MT2)-expressing cell line was made in the HEK
receptors belonging to the G-protein-coupled receptor (GPCR) 293-Gα15 host cells, which supported high levels of recombinant
superfamily are widely distributed in brain and peripheral nervous MT1 (or MT2) expression on the cell surface and contained high
systems of mammals,2-7 which are fascinating targets for drug levels of the promiscuous G protein Gα15 to enhance coupling of
discovery due to their multiply regulatory functions.8-10 MT1 the receptor to the calcium signaling pathway. Based on the Ca2+
receptor, generally expressed in the central nervous system and influx , the receptor channel pore could be controlled by agonists
many peripheral tissues,11 modulates neuronal firing. MT2 receptor using fluorimetric techniques, monitored by Ca2+-sensitive dyes.29
is observed in brain and lung, closely related to circadian rhythms of Test data for detecting changes in intracellular calcium was the
neuronal firing in the suprachiasmatic nucleus and dopamine transient calcium flux observed after activation of G-coupled
release in retina.12 In recent years, different types of MT1 and MT2 protein receptors .30 The details of Fluo-8 calcium assay were
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
a.State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR
China
，E-mail address: chenjj@mail.kib.ac.cn；Fax: + 86-0871-6522-7197；Tel:
+ 86-0871-6522-3265.
b.University of Chinese Academy of Sciences, Beijing 100049, PR China
c.Footnotes relating to the title and/or authors should appear here. Electronic
Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Scheme 1. Reagents and conditions: (a) DMAP, DCC, CH2Cl2, r.t., 60-70% for
compounds 1a-1x. (b) PCC, CH2Cl2, r.t., 95% for compound 2. (c) CH3CN,
reflux, 50-65% for compounds 1m.
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J. Name., 2013, 00, 1-3 | 1
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listed.31 Our previous bio-assay evaluation suggested that tropine
exhibited moderate agonistic activities on MT1 and MT2 receptors activities on MT1 and MT2 receptors with the values of 90.50% and
with the values of 90.50% and 77.48% at 1mM. Thus, a series of 77.48% at 1mM. When the hydroxyl group was changed to be a
tropine derivatives were synthesized for developing new MT carbonyl group, derivative 2 obviously reduced activities on MT1
receptors agonists. The preliminary structure-activity relationships and MT2 receptors with the values of 18.42% and 26.73% at 1mM
(SARs) were also discussed based on the bioassay on HEK293 cells in respectively, suggesting C=O group was unfavorable activity for
Journal Name
As shown in Table 2, tropine displayed moderate agonistic
View Article Online
DOI: 10.1039/C6RA06748D
vitro.
maintaining activities (Figure 2). In an effort to gain more
With an objective to obtain target compounds which have information of the SARs of tropine derivatives, the esterified
agonistic activities on HEK293 cells in vitro, the synthetic strategies products were further obtained.
for target compounds are flexible. Tropine was reacted with various
organic acids by Steglich esterification in the mixed solution of a
Table 1 Calculated physicochemical properties and predicted toxicity of
catalytic amount of 4-dimethylaminopyridine (DMAP), dehydrating
compounds 2 and 1a-1x.
agent N,N-dicyclohexylcarbodiimide (DCC) and anhydrous CH2Cl2 to
generate compounds 1a-1f and 1i-1l (Scheme 1).
Toxicity risksd
Clog pa
TPSAb (Å2)
20.31
logSc
-1.26
M/T/I/R
L/L/L/L
The results of preliminary bioassay on MT1 receptor showed that
Compd
introduction of
a
benzoyl group and a cinnamoyloxy group
2
0.65
200
190
180
170
160
150
140
130
120
110
100
90
1a
1b
1c
1d
1e
1f
0.99
1.89
2.12
2.57
6.44
2.43
3.04
3.16
2.25
2.76
2.46
1.42
2.32
3.10
3.10
2.86
2.86
29.54
29.54
29.54
29.54
29.54
29.54
29.54
29.54
29.54
29.54
38.77
67.87
32.78
29.54
29.54
29.54
29.54
-1.62
-2.16
-2.56
-2.92
-4.86
-2.79
-3.52
-3.62
-2.05
-3.16
-2.84
-2.49
-2.24
-3.50
-3.50
-3.47
-3.47
L/L/L/L
L/L/L/L
M/H/L/H
L/L/H/L
L/L/L/L
L/L/L/L
L/L/L/L
L/L/L/L
L/L/H/L
L/L/H/L
L/L/H/L
L/L/L/L
L/L/L/L
L/L/L/L
L/L/L/L
L/L/L/M
L/L/L/L
80
70
50
60
40
30
10
20
0
2
1i
1l
1f
1j
1a
1c
1e
1b
1d
1k
1m
Compounds were tested at the concentration of 1.00 mM. The agonistic activities
1g
1h
1i
̅
expressed as ± SD (n = 3) were obtained by comparing to the highest agonistic
activity that was achieved for melatonin at the highest concentration and was set as
100%.
Figure 1. Agonistic activities of derivatives 2, 1a-1f and 1i-1m on MT1
1j
receptor.
1k
1l
improved agonistic activity compared with that of tropine (Figure
1), and thus, derivatives 1g-1h and 1n-1x 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, HRMS and physicochemical
properties (including clog P – calculated logarithm of partition
coefficient between n-octanol and water, TPSA-polar surface area,
logS – a unit stripped logarithm of the solubility measured in
mol/liter and toxicity profiles (including mutagenic effect,
tumorigenic effect, irritating effect and reproductive effect).
Physicochemical properties of these compounds were calculated
and predicted using OSIRIS Property Explorer software at URL
1m
1n
10
1p
1q
1r
1s
2.86
3.37
2.69
3.97
2.62
29.54
29.54
38.77
29.54
48.00
-3.47
-3.89
-3.17
-4.63
-3.19
L/L/L/L
L/L/L/L
L/L/H/M
L/L/L/L
L/L/L/L
1t
1w
1x
a clog P, calculated logarithm of partition coefficient between n-octanol and water. b
TPSA, polar surface area. c S, Solubility. dToxicity risks: M, mutagenic effect; T,
tumorigenic effect; I, irritating effect; R, reproductive effect; L, low; M,medium; H,
high.
http://www.organic-chemistry.org/prog/peo/.32,
data were shown in Table 1.
The calculated
33
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DOI: 10.1039/C6RA06748D
Figure 2. Structure–activity relationships of tropane derivatives.
Most of esterified derivatives showed attractively agonistic exhibited increased agonistic activity on MT2 receptor. Derivative 1c
activities on MT1 and MT2 receptors (Table 2). The acetylated with a chlorinated group at the C-4 position of the butyryl group
showed similar agonistic activity with derivative 1b. Derivative 1d
Table 2. Agonistic activities of tropine derivatives (General molecular formula 1,
with a 2-methyl-valeryl group owned an approximately 2-fold
agonistic activity on MT2 receptor and similar agonistic activity on
Figure 3) on melatonin receptorsa.
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(Figure 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
Agonistic activities (%)
b
c
Comp.
R
MT1
MT2
Ago.
2
Tropine
1a
-
-
H
100.00 ± 3.54
18.42 ± 5.72
90.50 ± 3.33
69.51 ± 3.43
21.63 ± 3.70
-3.06 ± 0.45
73.52 ± 4.62
4.20 ± 1.26
100.00± 2.00
26.73 ± 1.84
77.48 ± 5.67
Acetyl
98.45 ± 4.35
1b
1c
1d
Butyryl
149.97 ± 33.86
106.34 ± 8.71
141.05 ± 13.91
88.60 ± 14.28
219.61 ± 7.28
127.05 ± 15.81
88.69 ± 2.03
4-Chloro butyryl
2-Methylvaleryl
Myristoyl
1e
1f
1g
1h
Benzoyl
194.22 ± 3.15
100.44 ± 3.50
47.65 ± 1.74
163.13 ± 5.70
197.18 ± 14.55
-2.55 ± 0.24
96.37 ± 2.97
173.02 ± 2.35
4-Chlorlbenzoyl
4-Bromoxybenzoyl
Senecioyl
Cinnamoyl
3-Phenoxy-propionyl
Boc-glycyl
1i
1j
1k
173.01 ± 50.85
215.67 ± 14.01
193.87 ± 7.35
95.64 ± 1.91
1l
1m
4-(N-piperidyl)butyryl
217.84 ± 12.70
a Agomelatine was tested at the concentration of 3.33 μM.and Other compounds were
b
̅
tested at the concentration of 1.00 mM. The agonistic activities expressed as ± SD (n = 3)
were obtained by comparing to the highest agonistic activity that was achieved for melatonin
at the highest concentration and was set as 100%.
derivative 1a displayed similar agonistic activities compared with
tropine. Derivative 1b, with a butyryl group at C-3 position,
Figure 3. General molecular formula 1 and 2 .
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activities than derivatives 1g and 1h with para-substituted at the fluorinated analogue at para-position of phenyl ring exhibited
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phenyl ring. Agonistic activities of derivative 1i with a conjugate higher activity than derivative 1j on MT2DOreI:c1e0p.1t0o3r9./CC6hRloAr0i6n7a4t8eDd
double bond was two times higher than that of the tropine on both derivative (1s) at meta-position of phenyl ring displayed slightly
receptors. Cinnamoyloxy substituted derivatives 1j with a conjugate lower activity than derivative 1j on both receptors. Derivative 1t
double bond exhibited excellent agonistic activities on both with a methoxy group at the C-3 position of the phenyl ring showed
receptors(Figure 2), which encouraged us to further explore similar agonistic activities on both receptors compared with
derivatives 1o-1x. Derivative 1k with a phenoxylpropionyl group derivative 1j. The dichloro substituted analogue (1w) and the
showed a 3-fold agonistic activity on MT2 receptor but no activity
on MT1 receptor. Derivative 1i with a N-(tert-butoxycarbonyl)-
Compound1rDose Response toMT
Compound1rDose Response toMT
1
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.
2
180
150
120
90
240
210
180
150
120
90
60
60
30
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-1x, 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 analogoues (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
30
0
0
100
101
102
103
101
102
103
MT[μΜ]
MT[μΜ]
EC50
245.4
EC50
199.6
Figure 4. The dose-dependent effects of derivative 1r on MT1 and MT2 receptors.
dimethoxy substituted analogue (1x) resulted in remarkably
reduced activity on both receptors compared with derivative 1j.
The dose-response curves for the most potent derivative 1r was
investigated to provide EC50 values of 0.20 and 0.24 mΜ on MT1 and
MT2 receptors, respectively (Figure 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.
Table 3. Agonistic activities of cinnamoyloxytropane derivatives (General
molecular formula 2 , Figure 3) on melatonin receptorsa.
Conclusions
Agonistic activities (%)
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.
b
c
Comp.
Ago.
1n
R1
-
R2
-
R3
-
MT1
MT2
100.00 ± 3.54
158.60 ± 9.63
145.56 ± 2.76
142.46 ± 6.25
160.17 ± 9.02
190.04 ± 9.13
125.35 ± 2.64
146.35 ± 10.32
56.65 ± 8.75
63.00 ± 6.26
100.00± 2.00
200.66 ± 4.50
223.44 ± 10.94
177.57 ± 12.79
196.72 ± 3.75
255.23 ± 10.01
183.36 ± 2.35
193.24 ± 8.02
36.56 ± 10.02
30.53 ± 2.81
Me
H
H
H
1o
H
Me
H
1p
F
H
1q
H
F
H
Author contributions
1r
H
H
F
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.
1s
H
Cl
H
H
1t
H
OMe
Cl
1w
1x
Cl
H
H
OMe
OMe
a Agomelatine was tested at the concentration of 3.33 μM.and Other compounds were
b
̅
tested at the concentration of 1.00 mM. The agonistic activities expressed as ± SD (n =
3) were obtained by comparing to the highest agonistic activity that was achieved for
melatonin at the highest concentration and was set as 100%.
4 | J. Name., 2012, 00, 1-3
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Firestinea, M. A. Melan. Life Sci. 2003, 72, 2183.
Acknowledgment
29. J. Walstab, S. Combrink, M. Brüss, M.DGOöI:t1h0e.1r0t,3V9Bie/.Cw6NARriteAics0lel6eO7r4n,l8iHnDe.
Bönisch. Anal. Biochem. 2007, 368, 185.
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.
30. P. Gribbon, C. Chambers, K. Palo, J. Kupper, J. Mueller, A.
Sewing. J. Biomol. Screen. 2006 , 11, 511.
31. In vitro agonist activity: The Fluo-8 Calcium Assay could
provide a fast, simple and reliable fluorescence-based assay for
detecting changes in intracellular calcium. HEK293 cell lines
stably expressing the human melatonin MT1 or MT2 receptor
was grown in Dubecco′s modified Eagle′s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), and cultured
with 95% O2/5% CO2 at 37℃. The cells were seeded in a
Matrigel coated 96-well black plate with a plating volume of
100 μL / well at a density of 4×104/well, and incubated in CO2
incubator (Thermo Forma 3310, US) for overnight. Then the
cells were dyed by HDB Wash Free Calcium Assay Kit, and
placed in CO2 incubator for 1h. Tested compounds and positive
drug were dissolved in 10 μL dimethyl sulfoxide (DMSO) and
990 μL HBSS Buffer respectively, and extracted a plating
volume of 100 μL/well in a Matrigel coated 96-well plate. Two
96-well plates were put into Flexstation 3 Benchtop Multi-
Mode Microplate Reader (Molecular Devices, Sunnyvale,
California, USA). The absorption values were readed by
Flexstation 3 Benchtop Multi-Mode Microplate Reader at
room temperature with wavelength (Excitation: 485 nm;
Emissiom: 525 nm; Emission cut-off: 515 nm ). The agonistic
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DOI: 10.1039/C6RA06748D
HEK293 cells in vitro, MT1: EC50 = 0.20 μM; MT2: EC50 = 0.24 μM.