Targeting AR with coumarins
Maria João Matos et al.
Table 1 Affinity (Ki), for binding to human A1, A2A and A3 adenosine receptors expressed in CHO cells, of coumarins 3–9, in radioligand binding assays
Ki (nM)
Selectivity
Compound
hA1
hA2A
hA3
hA1/hA3
hA2A/hA3
3
4
5
6
7
8
9
53 900 (35 900–81 100)
>100 000
25 000 (21 600–29 000)
7 760 (4 070–14 800)
>100 000
>30 000
>100 000
>30 000
36 600 (20 500–65 500)
7 160 (5 700–9 000)
>100 000
27 800 (22 600–34 300)
13 700 (12 000–15 600)
5 500 (3 490–8 670)
>100 000
7.5
–
>4.2
–
>0.9
0.57
>18.2
–
>1.1
2.7
>18.2
–
>100 000
>100 000
>30 000
>100 000
>100 000
>100 000
–
–
These results are the average of three experiments.
4-Hydroxy-3-(isobutylcarbamate)coumarin (8): Yield
Biological assays
1
77%. Mp 103–104°C. H NMR dppm (CDCl3-300 MHz):
0.96 (6H, d, J = 6.6 Hz, 2xCH3), 2.00 (1H, s, CH), 4.00 (2H,
d, J = 7.0 Hz, CH2), 7.28–7.37 (2H, m, H-6, H-8), 7.52–7.62
(1H, m, H-7), 7.93 (1H, dd, J = 7.9, 2.0 Hz, H-5), 7.48 (1H, s,
NH), 12.38 (1H, s, OH). 13C NMR dppm (CDCl3-75 MHz):
18.9, 27.9, 73.6, 104.0, 116.2, 117.1, 123.8, 124.6, 131.2,
150.1, 150.3, 157.1, 160.6. MS (ESI): 278 (7), 277 (M+, 40),
203 (27), 177 (100), 148 (15), 121 (65), 88 (18), 57 (50).
Anal. Calcd for C14H15NO5: C, 60.64; H, 5.45. Found: C,
60.60; H, 5.41.
The affinity of compounds 3–9 for the human AR subtypes
hA1, hA2A, hA3, was determined with radioligand competi-
tion experiments in Chinese hamster ovary (CHO) cells
that were stably transfected with the individual receptor
subtypes.[36,37] The radioligands used were 1 nm (2R,
3R,4S,5R)-2-(2-Chloro-6-cyclopentylamino-purin-9-yl)-5-
hydroxymethyl-tetrahydro-3,4-diol ([3H]CCPA) for hA1,
10 nm (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-b-D-
ribofuronamide) ([3H]NECA) for hA2A, and 1 nm 2-(1-
hexynyl)-N6-methyladenosine [3H] ([3H]HEMADO) for
hA3 receptors. The results were expressed as Kis (dissociation
constants), which were calculated with the program SCT-
FIT.[38] Duetothelackof asuitableradioligandfor hA2B recep-
tors,thepotencyof antagonistsatthehA2B receptor(expressed
on CHO cells) was determined by inhibition of NECA-
stimulatedadenylylcyclaseactivity.[36]
3-(Ethylcarbamate)-4-hydroxycoumarin (9): Yield 85%.
1
Mp 152–153°C. H NMR dppm (CDCl3-300 MHz): 1.35
(3H, s, CH3), 4.28 (2H, s, CH2), 7.24–7.35 (4H, m, H-5, H-6,
H-7, H-8), 7.93 (1H, s, NH), 12.35 (1H, s, OH). 13C NMR
dppm (CDCl3-75 MHz): 14.4, 63.7, 104.1, 116.2, 117.1,
123.8, 124.6, 131.2, 150.1, 150.3, 157.0, 160.6. MS (ESI): 250
(10), 249 (M+, 43), 203 (59), 177 (30), 148 (41), 121 (61), 65
(13). Anal. Calcd for C12H11NO5: C, 57.83; H, 4.45. Found:
C, 57.85; H, 4.47.
Statistical analysis
Ki values (Table 1) are reported as geometric means of three
independent experiments with each tested concentration of
compound measured in duplicate.As an interval estimate for
the dissociation constants,95% confidence intervals are given
in parentheses.
Biological results
The data from radioligand binding experiments for
compounds 3–9 are summarized in Table 1. Details for
pharmacological experiments are described in Materials and
Methods and in previous work.[36–38]
Results
Structural identification
Discussion
3-(Isobutylcarbamate)coumarin (7): Yield 78%. Mp
92–93°C. H NMR dppm (CDCl3-300 MHz): 1.39 (6H, d,
A novel coumarin series possessing a common planar
N-C = O framework, represented by an amidic or a carbam-
ate group at position 3,were studied for their ability to bind to
ARs. In addition, the presence or the absence of a hydroxyl
group at position 4 was also explored in these preliminary
studies.
The experimental results (Table 1) reveal that none of the
4-hydroxy derivatives (compounds 4, 8 and 9) have binding
affinity for any AR subtype. The presence of a hydroxyl func-
tion at position 4 of the coumarin scaffold is not tolerated,
1
J = 6.8 Hz, 2xCH3), 2.38 (1H, s, CH), 4.39 (2H, d, J = 6.7 Hz,
CH2), 7.69 (1H, s, H-4), 7.70–7.74 (2H, m, H-6, H-8), 7.80
(1H, dd, J = 7.3, 1.8 Hz, H-7), 7.88 (1H, dd, J = 7.6, 1.8 Hz,
H-5), 8.7 (1H, s, NH). 13C NMR dppm (CDCl3-75 MHz):
19.6, 28.5, 72.6, 116.9, 120.5, 121.4, 125.0, 125.7, 128.1, 129.8,
150.2, 154.1, 159.1. MS (ESI): 262 (7), 261 (M+, 42), 188 (23),
161 (70), 133 (28), 57 (41). Anal. Calcd for C14H15NO4: C,
64.36; H, 5.79. Found: C, 64.39; H, 5.83.
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 30–34
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