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Figure 6. Dose–response curve for compounds 6 and 8 in the PDK4 assay showing
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24. c log P 4.3 is a predicted octanol/water partition coefficient from Daylight
Chemical Information Systems and BioByte Corp.
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binding domain of human PPARd fused to the GAL4 DNA binding domain. The
primary PPARd screening assay contained the most common sequence of
PPARD (NM_006238). The cell stably expresses a luciferase gene under the
control of a GAL4 promoter. Compounds which bind and activate the PPARd/
GAL4 fusion promote luciferase production, which was quantified by
measuring luminescence generated from a luciferase substrate. GW501516 1
was used as reference compound and the intrinsic activity of 1 was set to 100%.
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about 10% of the total judged by LC–MS response, leaving over 80%
unchanged parent. All of these six metabolites were derived from
chemical modification of the azepine ring, three with loss of 2H,
and three hydroxylated derivatives, one of which was the glucuro-
nide. Furthermore, the compounds showed no cardiac QT liability
in vitro and exhibited excellent glutathione stability (pH 7.4),
chemical (pH 2, 7.4 and 11) and plasma stability.
Compound 10, slightly higher in lipophilicity, was yet more
potent in the PPARd agonist assay, with an EC50 value of 0.9 nM.
Additionally, this showed excellent PPAR selectivity: 4500-fold
selectivity against PPARa and over 20,000-fold over PPARc.
Activity in a relevant human biological effect assay (primary
human myotubes)35 has been determined for two key compounds
in this series, 6 and 8, with GW501516 (1) included as a reference
compound (Fig. 6). This assay was configured to measure upregu-
lation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA.
PDK4 is a mitochondrial enzyme, which inhibits glucose oxidation
via inactivation of the pyruvate dehydrogenase complex, thereby
increasing fatty acid oxidation. For compound 6, potency in this as-
say (EC50 = 69 nM) was consistent with the potency determined in
the hPPARd reporter assay (EC50 = 25 nM). Similarly, compound 8,
displayed a potency of 2 nM in the biological effect assay, and
3.2 nM in the reporter assay.
Some exploratory chemistry was conducted around modifica-
tions to the acetamide linker, but generally proved unsuccessful
in delivering compound improvements. For example, the urea
functionality in 16 (Table 1) was not tolerated and the ether com-
pounds 17 and 18 were only weak agonists at PPARd, showing that
the acetamide linker is intolerant of changes.
In summary, a series of small molecule PPARd agonists has been
found that displays excellent selectivity over the PPARa and PPARc
subtypes. Compounds 6 and 8 demonstrated efficacy in upregula-
tion of PDK4 in human primary myotubes, whilst compound 8
showed a good pharmacokinetic profile in the rat.
27. Selected experimental and analytical information: Compound 5: (2-(3-(2-(2,4-
dichlorophenylamino)-2-oxoethyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-
yloxy)acetic acid): To
a stirred solution of tert-butyl-2-(3-(2-(2,4-dichlo-
rophenylamino)-2-oxoethyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy)
acetic acid (280 mg, 0.58 mmol) in dichloromethane (3 mL) at rt was added
TFA (1.5 mL), and the solution was stirred at rt for 2 h. The mixture was
concentrated in vacuo and was subjected to mass-directed reverse-phase HPLC
Acknowledgement
purification (0.1% NH3(aq)/MeOH) on
a
GeminiÒ column NX C18 (30 ꢀ
100 mm) to provide 5 (58 mg, 24%) as a white solid. Purity 98.3% @ 220 nm;
LRMS (APCI) m/z [M+H]+ = 423, 425; [MꢁH]ꢁ = 421, 423; HRMS m/z
[M+H]+ = 423.0855, calcd 423.0873, (err. 4.25 ppm); 1H NMR (399.8 MHz,
DMSO) d 10.18 (s, 1H), 8.33 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 2.3 Hz, 1H), 7.46
(dd, J = 8.8, 2.4 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.65 (d, J = 2.6 Hz, 1H), 6.56
(dd, J = 8.2, 2.6 Hz, 1H), 4.30 (s, 2H), 3.27 (s, 2H), 2.91–2.84 (m, 4H), 2.74–2.65
(m, 4H), acid OH resonance absent; 13C NMR (125.8 MHz, DMSO) d 170.4,
169.0, 156.7, 142.3, 133.6, 133.1, 129.5, 128.6, 127.9, 127.7, 123.5, 121.9, 115.2,
111.2, 65.9, 62.1, 55.9, 55.5, 36.3, 35.2; melting point 205–210 °C (dec).
Elemental Anal. Calcd for C20H20Cl2N2O4ꢂ0.55NH3, 0.45H2O: C, 54.50; H, 5.16; N
8.10. Found: C, 54.47; H, 5.06; N, 8.12.
Compound (R)-8: (2-(2-(1-(2,4-dichlorophenylamino)-1-oxobutan-2-yl)-2,3,
4,5-tetrahydro-1H-benzo[c]azepin-8-yloxy)acetic acid): To a stirred solution
of 35 (methyl 2-(2-(1-(2,4-dichlorophenylamino)-1-oxobutan-2-yl)-2,3,4,5-
tetrahydro-1H-benzo[c]azepin-8-yloxy)acetate, 63 mg, 0.14 mmol)28 in THF
(3 mL) and water (1.5 mL) was added lithium hydroxide (16 mg, 0.68 mmol).
This solution was stirred at rt for 18 h. Acetic acid (six drops) was added to the
The authors gratefully acknowledge the technical support of
Danny Wang for the synthesis of the amine oxalate salts 28 and 29.
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