A series of analogues with alicyclic side chains were
synthesized to determine tolerance of steric bulk at position 6 of
the 4-oxo-1,4-dihydro-quinoline-2-carboxylic acid. Cyclopropyl-
methoxy analogue 50 in the carboxylic acid series showed more
than 10 times lower ETA receptor antagonist activity and
selectivity compared to the corresponding open chain i-butyl
analogue 46. Reduction in ETA receptor antagonist activity and
selectivity of cyclopropylmethoxy analogue 60 (IC50 = 78.4 nM)
of the tetrazole series compared to the i-butyl compound 56 were
found to parallel the effects of the carboxylic acid derivatives 50
and 46. This considerable reduction in antagonist activity and
receptor selectivity could be due to the fact that C-C bonds in
cyclopropane have more π character than σ character, resulting in
cyclopropylmethoxy substituent acting like an unsaturated
allyloxy substituent at position 6 of the quinolone ring. This
behavior resembles the ETA receptor binding nature of the
unsaturated allyloxy derivatives 47 and 57 compared to the
corresponding saturated n-propoxy derivatives 1 and 54.
ETA receptor antagonist activity was found to decrease by 3-
fold with the increment of each methylene group to increase the
ring size of the alicyclic analogue 50 to obtain
cyclobutylmethoxy analogue 51 and cyclopentylmethoxy 52 in
the carboxylic acid series. Compounds 51 and 52 also exhibited
poor ETA-selectivity. A similar reduction of 3-fold in ETA
receptor antagonist activity and poor ETA-selectivity was
observed with an increase in the ring size of cyclopropylmethoxy
analogue 60 in the tetrazole series to yield cyclobutylmethoxy
analogue 61 and cyclopentylmethoxy 62.
Scheme 5. Synthesis of compounds 41-62. (a) R1-X, DMF, NaH, K2CO3,
0 °C to rt. (b) & (c) 6N KOH, Ethanol, Reflux.
Branched alkyl, i-butoxy analogue 56 (IC50 = 6.2 nM) of the
tetrazole series had 5-fold lower ETA receptor antagonist activity
and selectivity in comparison with straight chain analogue 55.
Elimination of the alkyl chain entirely to obtain 6-OH analogue
41 (IC50 = 37.01 nM) in the carboxylic acid series led to 10-fold
decrease in ETA receptor antagonism and was accompanied by
1.6 times decrease in ETA-selectivity compared to the most
potent analogue 44 in this series. Removal of the alkyl chain in
the tetrazole series to obtain analogue 42 caused the reduction of
ETA receptor antagonist activity by 40-fold and selectivity by 1.8
times compared to the most potent compound 54 in this series.
In conclusion, a series of endothelin receptor antagonists was
synthesized and evaluated for their ETA and ETB receptor
antagonist activity and selectivity using GeneBLAzer® FRET
assay technology. The structure activity relationship deduced for
the carboxylic acid series was found to parallel that of the
tetrazole series. The n-propoxy analogue 54 in the tetrazole series
was found to be the most potent and selective ETA receptor
antagonist with an IC50 value of 0.8 nM and an ETA-selectivity of
1004. Target compounds with ethoxy, n-propoxy, n-butoxy, i-
butoxy substituents in both series were found to have similar or
better activity than the positive control BQ-123. These results
suggest that short chain saturated alkoxy groups at position 6 of
the 4-oxo-1,4-dihydro-quinoline-2-carboxylic acid impart good
ETA receptor antagonism and selectivity over ETB receptor.
Tetrazole 54 exhibits similar or better ETA receptor antagonist
activity as compared to the clinically approved endothelin
receptor antagonists for PAH (Figure 1) with ETA-selectivity
profile similar to that of the most recently approved drug,
macitentan.
Replacement of the saturated n-propoxy group in compound
44 with the unsaturated allyloxy group to obtain compound 47
resulted in 10-fold decrease in ETA receptor antagonism and
more than 20-fold decrease in ETA-selectivity. Analogous
unsaturated allyloxy compound 57 in the tetrazole series was
found to have diminished ETA receptor antagonist activity by 60-
fold and ETA-selectivity by 50-fold compared to n-propoxy
compound 54. This suggests that a saturated alkyl chain at
position 6 of 4-oxo-1,4-dihydro-quinoline-2-carboxylic acid is
beneficial for ETA receptor antagonist activity. Also, when the n-
propoxy chain in 44 is replaced by isosteric methoxymethyoxy
chain in the carboxylic acid series to obtain analogue 48, there is
6 fold loss of ETA receptor antagonism and 15 fold loss of ETA-
selectivity.
Acknowledgments
The authors thank Dr. Vladimir Poltoratsky and Zhihui Xiao
for their help with the in vitro FRET assay. Support for this
research was provided by Saint John’s University.
A similar reduction in ETA-selectivity for methoxymethoxy
analogue 58 in the tetrazole series was observed although the
ETA receptor antagonist activity reduced by nearly 20-fold.
Replacement of the terminal methyl group of the n-propoxy side
chain of 44 in the carboxylic acid series with an amino group led
to compound 49 which exhibited 85-fold reduction in ETA
receptor antagonism. ETA-selectivity also declined drastically
from 683 to 4 compared to 44. The 2-aminoethoxy analogue 59
in the tetrazole series resulted in more than 450-fold reduction in
ETA receptor antagonist activity and a virtual loss of ETA-
selectivity compared to 54. This suggests that substitution of an
amino group in place of the terminal methyl group of the n-
propoxy side chain is detrimental for ETA receptor antagonism
and selectivity.