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4289
Table 3. Evaluation of active quinolinones for inhibition of activity of CYP isozymes17—IC50 values (lM)
Compound
1A2/CEC
2C9/7-MFC
2C19/CEC
2D6/AMMC
3A4/BFC
3A4/BzRES
1
15
17
19
30
>40
1.7
3.9
16
5.2
24
35
23
8.1
13
>100
18
50
>100
>100
8.2
12
>100
>100
26
30
19
13
generally amenable to parallel synthesis for rapid gener-
ation of analogs for evaluation. For the synthesis of the
three classes of analogs, the key intermediate turned out
to be the tert-butyldiphenylsilyl (TBDPS) ether (9) ob-
tained by standard silylation procedure.6c Alkylation
of this silyl derivative with iodoacetonitrile resulted in
the formation of O-alkyl derivative 10 and N-alkyl
derivative 11 in 9:1 ratio (35% yield), along with N,O-
dialkyl derivative 12 (41% yield). These silyl-protected
cyanomethyl derivatives were separated by silica gel
chromatography and used for subsequent derivatization
(Scheme 1).
Of the two acidic sites present in the quinolinone moiety,
the phenolic OH is important for activity as maxi-K
opener. Modification of the second N1 acidic site in
the quinolinones can result in disruption of recognition
by CYP2C9 without loss of activity as in maxi-K
opener.
In conclusion, we have demonstrated that modification
of the acidic site in the quinolinone 1 can be used as
an approach to overcome the CYP2C9 enzyme inhibi-
tion while maintaining maxi-K activity.
Deprotection of protecting silyl and acetyl groups from
nitriles 10 and 13 gave nitriles 14 and 15 (ꢀ72% and 46%
yield, respectively). Amides 16 and 17 were obtained
from the corresponding nitriles by alkaline peroxide-
mediated hydrolysis13 and desilylation in an overall
yield of 42%. Elaboration of nitriles 10 and 13 to the
corresponding tetrazoles 18 and 19 occurred by the
cycloaddition of azide ion in DMF.14 Interestingly, this
process also resulted in concomitant desilylation. The
synthesis of amidines 20 and 21 was performed via the
corresponding methyl imidates.15
References and notes
1. (a) Gribkoff, V. K.; Dworetzky, S. I.; Starrett, J. E., Jr.
The Neuroscientist 2001, 7, 166; (b) Shieh, C.; Coghlan,
M.; Sullivan, J. P.; Gopalakrishnan, M. Pharmacol. Rev.
2000, 52, 557.
2. Clark, A. G.; Booth, S. E.; Morrow, J. A. Exp. Opin.
Therapeut. Patents 2003, 13, 23.
3. Hewawasam, P.; Erway, M.; Moon, S. L.; Knipe, J.;
Weiner, H.; Boissard, C. G.; Post-Munson, D. G.; Gao,
Q.; Huang, S.; Gribkoff, V. K.; Meanwell, N. A. J. Med.
Chem. 2002, 45, 1487.
4. Tanaka, M.; Sasaki, Y.; Kimura, Y.; Fukui, T.; Hamada,
K.; Ukai, Y. BJU Int. 2003, 92, 1031.
5. Christ, G. J.; Rehman, J.; Day, N.; Salkoff, L.; Valcic, M.;
Geliebter, J. Am. J. Physiol. 1998, 275, 600.
The target compounds thus obtained were evaluated in
Xenopus laevis oocytes, expressing the cloned hSlo
maxi-K channel for their ability to open the maxi-K
channel.16 Table 2 illustrates the percent increase of hSlo
current obtained with the target compounds tested at
20 lM. In this assay, compounds demonstrating a value
P130% increase of measured maxi-K current are consid-
ered significant openers of the maxi-K channel.
6. (a) Hewawasam, P.; Fan, W.; Ding, M.; Flint, K.; Cook,
D.; Goggins, G. D.; Myers, R. A.; Gribkoff, V. K.;
Boissard, C. G.; Dworetzky, S. I.; Starrett, J. E., Jr.;
Lodge, N. J. J. Med. Chem. 2003, 46, 2819; (b) Hewawa-
sam, P.; Starrett, J. E., Jr. U.S. Patent 6184231 B1, 2001;
(c) Hewawasam, P.; Fan, W.; Knipe, J.; Moon, S. L.;
Boissard, C. G.; Gribkoff, V. K.; Starrett, J. E., Jr. Bioorg.
Med. Chem. Lett. 2002, 12, 1779; (d) Boy, K. M.;
Guernon, J. M.; Sit, S. Y.; Xie, K.; Hewawasam, P.;
Boissard, C. G.; Dworetzky, S. I.; Natale, J.; Gribkoff, V.
K.; Lodge, N. K.; Starrett, J. E., Jr. Bioorg. Med. Chem.
Lett. 2004, 14, 5089.
7. Guengerich, F. P.; Turvey, C. G. J. Pharmacol. Exp. Ther.
1991, 256, 1189.
8. Romkes, M.; Faletto, M. B.; Blaisdell, J. A.; Raucy, J. L.;
Goldstein, J. A. Biochemistry 1991, 30, 3247.
9. Mancy, A.; Broto, P.; Dijols, S.; Dansette, P. M.; Mansuy,
D. Biochemistry 1995, 34, 10365.
Modification of the phenolic OH by alkylation with sub-
stituents containing neutral or acidic or basic functional-
ities, as in analogs 14, 16, 18, and 20, led to diminished
activity as maxi-K openers, confirming the importance
of phenolic hydroxyl for channel opening ability.6b
The N1 position was also modified to introduce the same
substituents to obtain analogs 15, 17, 19, and 21. In this
series, nitrile 15, the weakly acidic amide 17, and the tet-
razole 19 were found to be potent maxi-K openers,
whereas the basic amidine 21 turned out to be inactive.
10. Rao, S.; Aoyama, R.; Trager, W. F.; Rettie, A.; Jones, J.
P. J. Med. Chem. 2000, 43, 2789.
Thus, the maxi-K channel tolerated neutral and weakly
acidic substituents on N1. Nitrile 15 displayed only 2-
fold reduction in CYP2C9 inhibition (IC50 = 3.9 lM)
compared to 1 (IC50 = 1.7 lM). However, compounds
17 and 19 showed 9- and 11-fold, respectively, reduced
inhibition of CYP2C9 (Table 3). Further evaluation
with other CYP isozymes showed that the profile across
a panel of CYP enzymes was generally better than 1,
with IC50 values for the common isoforms being in the
range of 8–100 lM.
11. ‘‘Applications and Theory Guide to pH-metric pKa and
logP Determination’’, Sirius Analytical Instruments Ltd,
and the references therein. 1995. Measurement of pKa was
made with a Sirius pION Model GLpKa instrument using
pH titration with potentiometric or spectrophotometric
detection. The test compound (2–10 mg) was dissolved in
10–20 ml aqueous solvent using 150 mM KCl to keep the
ionic strength constant. A potentiometric or spectropho-
tometric titration curve was generated automatically by
the GLpKa instrument. An organic cosolvent, methanol,