Journal of Medicinal Chemistry
ARTICLE
removed at the water-jet pump, and the still hot residue was
suspended in acetone (20 mL). The suspension was cooled to RT
with stirring and the solid was isolated by filtration. After washings
with acetone, the off-white solid was dried in vacuo to yield the
hydrochloride salt of 4-(5-chloro-2-benzimidazolyl)-3-(4-chlorophe-
nyl)butanoic acid (1a) (4.66 g, 60%). 1H NMR (500 MHz, DMSO-d6):
δ (ppm) = 2.72 (dd, J = 16.2, 8.6 Hz, 1H), 2.83 (dd, J = 16.2, 6.2 Hz, 1H),
3.43 (dd, J = 14.9, 9.2 Hz, 1H), 3.55 (dd, J = 14.9, 6.9 Hz, 1H), 3.83À3.89
(m, 1H), 7.30 (d, J= 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.48 (dd, J = 8.8,
1.9 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H). 13C NMR
and DEPT (125 MHz, DMSO-d6): δ (ppm) = 32.7 (CH2), 39.2 (CH),
39.6 (CH2), 113.6 (CH), 115.3 (CH), 125.5 (CH), 128.3 (2 CH), 129.1
(2 CH), 129.6 (C), 130.0 (C), 131.4 (C), 131.9 (C), 140.7 (C), 153.3
(C), 172.1 (CO). MS (+ESI): m/z = 349 (M + H).
(m, 5H), 7.50 (s, 1H), 7.60 (d, J = 7.3 Hz, 1H), 15.2 (br s, 2H). 13C NMR
and DEPT (125 MHz, DMSO-d6): δ (ppm) = 15.7 (CH3), 28.0 (CH2),
32.2 (CH2), 39.0 (CH), 39.7 (CH2), 111.8 (CH), 113.2 (CH), 125.8
(CH), 128.3 (2 CH), 128.6 (C), 129.1 (2 CH), 130.6 (C), 131.3 (C),
140.5 (C), 141.7 (C), 151.2 (C), 172.0 (CO). MS (+ESI): m/z = 343
(M + H).
Protein Kinases and Kinase Assays. PKCζ was purified as
glutathione S-transferase (GST)-fusion protein from HEK293 cells.
Cells were transiently transfected with 12.5 μg of an expression plasmid
coding for full length human PKCζ (pEBG-2T-PKCζ) mixed with
polyethylene imine (PEI, Polysciences Inc.) at a PEI/plasmid ratio of
10:1 (μL PEI stock solution of 1 mg/mL:μg DNA) per 14.5 cm diameter
Petri dish. At 36 h post-transfection, the cells were lysed in 1.5 mL of lysis
buffer (50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (by
mass) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium
fluoride, 10 mM sodium β-glycerophosphate, 0.27 M sucrose, 5 mM
sodium pyrophosphate, 0.1% (by volume) β-mercaptoethanol, and 1
tablet of protease inhibitor mixture (Roche) per 50 mL of buffer) per
dish. The lysates were cleared by centrifugation and incubated for 2 h at
4 °C with glutathione-Sepharose. After extensive washing of the beads
with lysis buffer containing 0.5 M NaCl, lysis buffer and finally in buffer
A (50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1% β- mercaptoethanol),
the GST-fusion protein was eluted using 500 μL of buffer supplemented
with 80 mM glutathione. Cell-free protein kinase activity assays were
performed essentially as described previously,36 using myelin basic
protein (MBP, Millipore) as an artificial substrate (0.2 mg/mL) and
started using 100 μM γ32P-ATP/Mg2+. Activator lipids of PKCζ such as
phosphatidyl serine were omitted in order to measure true allosteric
inhibition effects rather than the blocking of activation. Phosphorylated
peptides were spotted on P81 phosphocellulose paper (Whatman),
washed by diluted phosphoric acid, and incorporated 32P quantified in a
PhosphoImager. In a similar manner, PRK2 was expressed from pEBG-
2T-PRK2,46 SGK1 from pEBG-2T-SGK1-ΔN[Ser422Asp], PKBα from
pEBG-2T-PKBα[Ser473Asp],47 and PKCι from pEBG-2T-PKCι.
PDK1 and S6K1 were expressed as His-tag fusion proteins in Sf9 insect
cells using a baculovirus expression system from pFastBac-PDK1 and
pFastBac-S6K1-T2[Thr412Glu]. PKA was purchased from Sigma;
PKCα, IKKα, IKKβ, RIPK2, p38α, TAK1, and TBK1 were from
Millipore; PKCβ, θ, and δ were from ProQinase. The substrates were
T308tide (200 μM) for PDK1, Kemptide (100 μM) for PKA, and
Crosstide (100 μM) for SGK, PKB, S6K, and PRK2. Substrates used for
IKKα, IKKβ, RIPK2, p38α, TAK1, and TBK1 were described by Bain
et al.48 The activity assays for PKCα, β, θ, and δ were performed in the
presence of a PKC lipid activator mix (Millipore) using 3 μM of histone
H1 as the substrate.
Reporter Gene Assay. U937 cells were cultured in RPMI-1640
containing 10% FCS and penicillin/streptomycin. Transfection was
performed in 6-well plates at a density of 106 cells/mL (2 mL per well)
in the same medium without antibiotics. The transfection complex was
prepared by mixing the reporter gene plasmid (pGL4.32[luc2P/NF-kB-
RE/Hygro], Promega) with FuGene HD transfection reagent (Roche
Diagnostics) at a ratio of 1:6 (μg DNA:μL transfection reagent) in
RPMI-1640 medium according to the manufacturer's instructions. The
transfection mixture was added dropwise to the wells while gently
shaking, and the plates were incubated at 37 °C/5% CO2 for 6 h. The
medium was then exchanged to serum-free RPMI-1640/penicillin/
streptomycin without phenol red and the cells starved overnight. The
next day, test compounds dissolved in DMSO were pipetted into white
96-well plates (0.2 μL), followed by 100 μL of the transfected cells per
well. After 3 h at 37 °C/5% CO2, the NF-kB pathway was induced by the
addition of 50 ng/μL of TNFα to the wells (except uninduced controls).
The cells were incubated at 37 °C/5% CO2 for 2.5 h, then the luciferase
detection reagent was added (Bright-Glo, Promega, 100 μL per well)
3-(4-Chlorophenyl)-4-(5-methyl-2-benzimidazolyl)butanoic
Acid HCl (1f). 7-Methyl-3-phenyl-3,4-dihydropyrido[1,2-a]benzimidazol-
3
1(2H)-one and 8-Methyl-3-phenyl-3,4-dihydropyrido[1,2-a]benzimidazol-
1(2H)-one. The solution of 4-methyl-1,2-phenylenediamine (3f) (367 mg,
3 mmol) and 3-(4-chlorophenyl)glutaric anhydride (2a) (674 mg, 3mmol)
in THF (1 mL) was kept at RT for 0.5 h and subsequently decolorized with
activated carbon. After filtration over Celite and concentration of the filtrate,
the off-white residue was redissolved in acetic acid (5 mL). The solution was
heated to reflux overnight. After removal of the solvent in vacuo, the residue
was recrystallized from ethanol to afford a 1:1 mixture of the regioisomers
8-methyl-3-phenyl-3,4-dihydropyrido[1,2-a]benzimidazol-1(2H)-one(8a)
and 7-methyl-3-phenyl-3,4-dihydropyrido[1,2-a]benzimidazol-1(2H)-one
(8b) (250 mg, 27%) as colorless solid.
3-(4-Chlorophenyl)-4-(5-methyl-2-benzimidazolyl)butanoic Acid
HCl. The previous mixture (8a,b) (96 mg, 0.31 mmol) was converted
3
to 3-(4-chlorophenyl)-4-(5-methyl-2-benzimidazolyl)butanoic acid
HCl (1f) (84 mg, 74%) with a mixture of acetic acid (1 mL) and concd
3
HCl (0.5 mL) at reflux temperature for 1.5 h, with the removal of all
1
volatiles, and trituration of the residue with acetone. H NMR (500
MHz, DMSO-d6): δ (ppm) = 2.43 (s, 3H), 2.72 (dd, J = 16.2, 8.6 Hz,
1H), 2.82 (dd, J = 16.2, 6.2 Hz, 1H), 3.43 (dd, J = 15.0, 9.6 Hz, 1H), 3.56
(dd, J = 15.0, 6.7 Hz, 1H), 3.83À3.91 (m, 1H), 7.26À7.31 (m, 1H), 7.29
(d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.50 (s, 1H), 7.58 (d, J = 8.4
Hz, 1H).13C NMR and DEPT (125 MHz, DMSO-d6): δ (ppm) = 20.9
(CH3), 32.4 (CH2), 39.2 (CH), 39.7 (CH2), 113.1 (CH), 113.2 (CH),
126.8 (CH), 128.3 (2 CH), 128.6 (C), 129.1 (2 CH), 130.8 (C), 131.4
(C), 135.3 (C), 140.6 (C), 151.3 (C), 172.1 (CO). MS (+ESI): m/z =
329 (M + H).
3-(4-Chlorophenyl)-4-(5-ethyl-2-benzimidazolyl)butanoic
Acid HCl (1g). The solution of commercial 4-ethylaniline (3g)
3
(606 mg, 5 mmol) and 3-(4-chlorophenyl)glutaric anhydride (2a)
(1.12 g, 5 mmol) in 1,4-dioxane (3 mL) was stirred at 100 °C for 1 h.
The solvent was removed, and the oily residue was redissolved in acetic
acid (2 mL). With vigorous stirring at RT, fuming nitric acid (2 mL,
50 mmol) was added over the course of 1 min. After stirring at RT for 1 h,
the orange solution was quenched with ice water (50 mL). The yellow
gummy precipitate was separated by decantation, dried in vacuo, and
redissolved in acetic acid (10 mL) and one drop of concd HCl. The
solution was stirred at reflux temperature, while iron powder (1.4 g,
25 mmol) was added. After 1 h, a mixture of acetic acid (4 mL) and concd
HCl (14 mL) was added with caution (foaming). Stirring at reflux was
continued for 2 h to ensure the complete dissolution of the excess of iron.
Then, all volatiles were removed at the water-jet pump at an elevated
temperature. The residue was triturated with boiling acetone and filtered.
By the addition of concd HCl, the product was precipitated from the
concentrated acetone filtrate to give the hydrochloride salt of 3-(4-
chlorophenyl)-4-(5-ethyl-2-benzimidazolyl)butanoic acid (1g) (0.75 g,
1
40% calc. over 4 steps) as a beige colored solid. H NMR (500 MHz,
DMSO-d6): δ (ppm) = 1.19 (t, J = 6.3 Hz, 3H), 2.66À2.86 (m, 4H),
3.43À3.49 (m, 1H), 3.52À3.58 (m, 1H), 3.86À3.90 (m, 1H), 7.24À7.40
6721
dx.doi.org/10.1021/jm2005892 |J. Med. Chem. 2011, 54, 6714–6723