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M. Hammond et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4441–4445
diluted to
a final concentration of 0.5 nM, and SGK1, diluted to a final
ortho to the carboxylate might be accommodated; while that of 2
indicated that the a-carbon would also tolerate substitution with-
concentration of 1 nM in buffer containing 50 nM HEPES (pH 7.5), 1 mM
CHAPS, 10 nM MgCl2, and 1 mM DTT, were incubated for 15 min at RT.
Azaindole inhibitors were dissolved in DMSO and diluted into buffer for
concentration-response curve determination. The fluorescence signal was
measured using an Acquest (Molecular Devices; Sunnyvale, CA) at excitation
and emission wavelengths of 485 nm and 530 nm, respectively. SGK1
out disrupting binding. Boronic acid intermediates needed for the
synthesis of ortho-substituted analogs of 1 were accessed as shown
in Scheme 2.11 The starting 4-bromo-2-fluorobenzoic acid 10 was
treated with Grignard reagents to afford the 2-alkyl-4-bromoben-
zoic acid intermediates,12 which were converted to boronic acids
11 using a protocol involving metal-halogen exchange, trapping
with triisopropyl borate and hydrolysis with aqueous HCl. For
the latter approach, two boronates, 13 and 15 were prepared as
shown in Scheme 3. Phenylacetic acid boronate 12 was esterified
to its methyl ester 13,13 while the bromide 14 was converted to
its boronate ester 15.14 The boronic acids 11, 13, and 15 were then
utilized in the syntheses of target azaindoles 16–22 as detailed in
Scheme 1.15
inhibition IC50 was calculated from these data using GraphPad Prism
Software (GraphPad Software, San Diego, CA).
3
4. Nakhoul, N. L.; Hering-Smith, K. S.; Gambala, C. T.; Hamm, L. L. Am. J. Physiol.
1998, 275, F998.
5. Dutton, G. J. Glucuronidation of Drugs and Other Compounds; CRC Press: Boca
Raton, 1980. p 268.
6. Roberts, M. S.; Magnusson, B. M.; Burczynski, F. J.; Weiss, M. Clin.
Pharmacokinet. 2002, 41, 751.
7. Crystallographic data has been deposited in the PDB with deposition codes
3HDM for compound 1 and 3HDN for compound 2.
8. All target compounds met purity requirements of P95% as determined by
LCMS and/or 1H NMR.
9. Example azaindole preparation (1): Step 1: PdCl2(dppf) (1.04 g, 1.27 mmol,
The ortho-substituted analogs of 1 behaved very favorably in
the SGK1 in vitro assays, having improved inhibition potencies rel-
ative to 1 (Table 2, compounds 16–18). In general, increasing the
size of the substituent trended toward a corresponding increase
in isolated SGK1 inhibition and whole-cell potency. Phenylacetic
acid derivatives 19–22 also demonstrated good in vitro potencies.
These analogs appeared to be relatively insensitive to methyl sub-
0.05 equiv) was added in one portion to
a suspension of 5-bromo-1H-
pyrrolo[2,3-b]pyridine (5.00 g, 25.4 mmol, 1 equiv), phenylboronic acid (3.70 g,
30.5 mmol, 1.2 equiv), and K2CO3 (10.5 g, 76.1 mmol, 3 equiv) in 2.5:1 dioxane/
water (253 mL). The reaction mixture heated in an oil bath set to 80 °C. After
22.5 h, the reaction mixture was cooled to room temperature, acidified with 6 N
HCl, and partitioned between EtOAc and water. The mixture was filtered through
a pad of pressed Celite, and the filtrate layers were separated. The aqueous layer
was extracted with EtOAc and the combined organics were washed with brine,
dried over Na2SO4 and concentrated. The residue was purified using DOWEX
50WX2-400 ion exchange resin to provide 5-phenyl-1H-pyrrolo[2,3-b]pyridine
as a light brown solid (4.86 g, 99%). Step 2: Br2 (1.27 mL, 3.95 g, 24.7 mmol,
1 equiv) was added over a period of 35 min to a solution of 5-phenyl-1H-
pyrrolo[2,3-b]pyridine (4.8 g, 24.7 mmol, 1 equiv) in CHCl3 (247 mL). The
reaction mixture was stirred at room temperature for 15 min and then
concentrated in vacuo. The pale orange foam, 3-bromo-5-phenyl-1H-
pyrrolo[2,3-b]pyridine, was carried to the next reaction without further
purification. Step 3: (Bu4N)HSO4 (100 mg, catalytic) was added to a mixture of
3-bromo-5-phenyl-1H-pyrrolo[2,3-b]pyridine (24.7 mmol, 1 equiv) and p-
toluenesulfonyl chloride (5.65 g, 29.6 mmol, 1.2 equiv) in a bilayer of CH2Cl2
(308 mL) and 6 N NaOH (50 mL). After 1 h the reaction mixture was diluted with
water, filtered through a plug of Celite, and the filtrate layers separated. The
aqueous layer wasextracted withCH2Cl2. Thecombinedorganicsweredriedover
Na2SO4 and were concentrated. Purification of the residue by silica gel
chromatography (CH2Cl2 grading to 10% EtOAc/CH2Cl2) afforded 3-bromo-1-
[(4-methylphenyl)sulfonyl]-5-phenyl-1H-pyrrolo[2,3-b]pyridine (7.19 g, 68%
over two steps) as a tan solid. Step 4: PdCl2(dppf) complex with CH2Cl2
(246 mg, 0.30 mmol, 0,05 equiv) was added to a suspension of 3-bromo-1-[(4-
methylphenyl)sulfonyl]-5-phenyl-1H-pyrrolo[2,3-b]pyridine (2.57 g, 6.01 mmol,
1 equiv), 4-carboxyphenylboronic acid (1.2 g, 7.21 mmol, 1.2 equiv) and K2CO3
(2.49 g, 18.0 mmol, 3 equiv) in 2.5:1 dioxane/water (60 mL). The reaction mixture
was refluxed for 3 h and then cooled to rt and acidified with concentrated HCl. The
mixture was filtered through a pad of Celite, and the filtrate was partitioned
between EtOAc and water. The layers were separated, and the aqueous layer was
further extracted with EtOAc. The combined organics were dried over Na2SO4 and
were concentrated. The 4-[5-phenyl-1-(toluene-4-sulfonyl)-1H-pyrrolo[2,3-
b]pyridin-3-yl)-benzoic acid was used directly in the next step without further
purification. Step 5: Crude 4-[5-phenyl-1-(toluene-4-sulfonyl)-1H-pyrrolo[2,3-
b]pyridin-3-yl)-benzoicacid was takenupina mixtureof MeOH(50 mL) and2.5 N
NaOH (20 mL). The reaction mixture was heated at 50 °C for 30 min and then
acidified with concentrated HCl and partitioned between EtOAc and water. The
precipitate that formed wascollected byfiltrationandsetaside. Theaqueous layer
was further extracted with EtOAc, and the combined organics were dried over
anhydrous Na2SO4 and concentrated. The original precipitate was combined with
the residue from the organics, and the combination was stirred with 10% MeOH in
CH2Cl2 for 30 min. The insolubles were again collected by filtration and combined
with the first crop. This process was repeated once more to provide 4-(5-phenyl-
1H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid 1 (1.13 g, 60%). 1H NMR (400 MHz,
CD3OD) d 8.54 (s, 2H), 8.14 (d, 2H, J = 8 Hz), 7.87 (m, 3H), 7.73 (d, 2H, J = 8.8 Hz),
7.51 (t, 2H, J = 7.6 Hz), 7.40 (t, 1H, J = 7.2 Hz). LCMS (ES) m/z 315 [M+H]+.
stitution at the
a-position of the phenylacetic acid, with potency
against the isolated enzyme for compounds 2, 19, and 20 all within
twofold of each other. In addition, the whole-cell potency re-
mained unchanged across the set. Compound 21 was essentially
equipotent to its direct analog 1, and substitution of the phenyl
ring in the 5-position with a meta-cyano group (22) also did not
significantly change either isolated enzyme or whole-cell potency.
As hypothesized, steric obstruction of the carboxylate group
also resulted in significantly different rat pharmacokinetic proper-
ties (Table 2). The ortho-substituted azaindoles 16–18 showed sig-
nificantly lower plasma clearances, reduced volumes of
distribution, and shorter iv half-lives. The parameters for com-
pound 16 were sufficiently improved such that the DNAUC follow-
ing oral dosing was nearly fourfold improved over that for 1.
Although the rat oral plasma concentration versus time plot for
16 indicated a secondary input peak, it was significantly smaller
than that for 1. Interestingly, the DNAUC values for 17 and 18 were
very similar to that of 1 and significant secondary input peaks were
observed for both after oral dosing (data not shown). The most
marked improvement in rat oral exposure was observed for ana-
logs with substitution inserted in between the carboxylate and
the phenyl ring. Plasma clearance values for compounds 20 and
22 were at least threefold lower and the DNAUC values were as
much as 10-fold higher than those of 2 and no secondary input
peaks were observed in either of their oral exposure curves.
In summary, inhibitors of SGK1 were designed to address the
poor rat PK properties of lead azaindoles 1 and 2. Improvement
of oral exposure while maintaining SGK1 inhibition potency
proved to be the key challenge, as the carboxylate moiety was be-
lieved to be good for the latter but detrimental for the former. Rat
PK could be improved by addition of alkyl substituents ortho to the
10. Although the introduction of steric bulk near the site of glucuronidation would
appear to be a common strategy for reducing conjugate formation, extensive
literature searching turned up only a single report of such an observation:
Franklin, T. J.; Jacobs, V. N.; Jones, G.; Ple, P. Drug Metab. Dispos. 1997, 25, 367.
11. Example preparation of 4-(dihydroxyboranyl)-2-ethylbenzoic acid: Step 1: EtMgBr
(1 M in THF, 32.0 mL, 32.0 mmol) was added to a solution of 4-bromo-2-
fluorobenzoic acid (2 g, 9.13 mmol) in THF (15 mL) at 0 °C. After 4 h 25 mL of
2 N HCl was added slowly at 0 °C. EtOAc (30 mL) was added, and the layers
were separated. Aq NaOH (30 mL, 2.5 N) was added to the organic layer and the
mixture stirred for 30 min. The layers were separated, and the aqueous layer
was washed with EtOAc (10 mL) and then acidified to pH 2 with 6 N HCl. The
aqueous layer was extracted three times with EtOAc (30 mL), and the
combined organic extracts were dried over Na2SO4 and concentrated to give
4-bromo-2-ethylbenzoic acid as a white solid (0.575 g, 28%). Step 2: B(O-i-Pr)3
(3.52 mL, 15.3 mmol) was added to the acid (0.250 g, 1.09 mmol) in THF
(18 mL). The mixture was cooled to ꢀ78 °C and n-butyllithium (2.5 M in
hexanes, 6.1 mL, 15.3 mmol) was added over 10 min. The reaction temperature
carboxylate of 1 or by introducing geminal dimethyl groups
a to
the carboxylate of 2. Ultimately, several of the azaindoles de-
scribed were determined to meet the criteria set forth for in vivo
testing in pharmacological models.16
References and notes
1. Pearce, D.; Kleyman, T. R. J. Clin. Invest. 2007, 117, 592.
2. Wulff, P.; Vallon, V.; Huang, D. Y.; Volkl, H.; Yu, F.; Richter, K.; Jansen, M.;
Schlunz, M.; Klingel, K.; Loffing, J.; Kauselmann, G.; Bosl, M. R.; Lang, F.; Kuhl, D.
J. Clin. Invest. 2002, 110, 1263.
3. The SGK1 assay used fluorescence polarization to monitor the binding of test
compounds to the enzyme. The FP ligand, 30,60-diamino-N-[2-({3-[5-amino-6-
(1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-2-pyrazinyl]phenyl}amino)-2-
oxoethyl]-3-oxo-3H-spiro[2-benzofuran-1,90-xanthene]-5-carboxamide,