Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5873
was followed by TLC until no more starting compound 2 was
detectable. Compounds 3 partly crystallized from the solutions under
irradiation and were finally collected after reduction of the THF
solvent under cooling.
treatment of the P-gp inducible cell model with our inhibitors
4a and 4b did not result in detectable P-gp amounts as shown
in lanes 3 and 4.
Tetraethyl 3,9-Dibenzyl-6,12-bis(3-benzyloxyphenyl)-3,9-dia-
zahexacyclo[6.4.0.02.7.04.11.05.10]dodecane-1,5,7,11-tetracarboxy-
late (3f). Yield 0.275 g (46%); mp 169-178 °C; 1H NMR (CDCl3)
δ 7.35-7.17 (m, 20 H), 6.99 (t J ) 7.7 Hz, 2 H), 6.96 (s, 2 H),
6.80 (d J ) 7.7 Hz, 2 H), 6.72 (d J ) 7.7 Hz, 2 H), 4.88 (s, 4 H),
4.46 (s, 4 H), 4.27 (s, 2 H), 4.25 (s, 4 H), 3.97 (q J ) 7.0 Hz, 8 H),
0.97 (t J ) 7.0 Hz, 12 H); MS (ESI) m/z ) 996 (M + H+). Anal.
(C62H62N2O10) C, H, N.
Tetraethyl 3,9-Bis(3-benzyloxybenzyl)-6,12-diphenyl-3,9-dia-
zahexacyclo[6.4.0.02.7.04.11.05.10]dodecane-1,5,7,11-tetracarboxy-
late (3g). Yield 0.269 g (45%); mp 119-126 °C; 1H NMR (DMSO-
d6) δ 7.38-7.30 (m, 14 H), 7.22-7.06 (m, 10 H), 6.90-6.87 (m,
4 H), 4.77 (s, 4 H), 4.44 (s, 4 H), 4.27 (s, 2 H), 4.17 (s, 4 H), 3.92
(q J ) 7.0 Hz, 8 H), 0.93 (t J ) 7.0 Hz, 12 H); MS (ESI) m/z )
996 (M + H+). Anal. (C62H62N2O10) C, H, N.
General Procedure for the Formation of the Alcoholic Target
Compounds 4a-g. 3,9-Diazatetrasteranes 3 (1 equiv, 70 µM) were
dissolved in dried THF under warming. After syringing with argon,
the solution was stirred for 1 h at rt. After the mixture was cooled
to 8 °C, lithium aluminum hydride (16 equiv, 1.1 mmol) in a
solution in THF (1 M) was added dropwise under stirring, which
then continued for 3 h. Then the excess of lithium aluminum was
hydrolyzed with portions of ice-water. Extraction followed with
chloroform (3 × 60 mL). After drying over sodium sulfate and
filtration the solvent was removed in vacuum. The residual oil was
taken up in chloroform, and after addition of mixtures of diethyl
ether and petrol ether the target compounds 4 crystallized and where
recrystallized from methanol/water.
Conclusions
We developed novel P-gp inhibitors with mainly improved
activities up to 50-fold compared to verapamil. Methoxy
functions as hydrogen bond acceptor functions in compounds
4b and 4c improved activities in dependence of their molecular
presentation within the aromatic residues. Benzyloxy functions
instead of the methoxy functions mainly led to increased
activities so that the increased lipophilicity was found favorable.
Highest activities were found for meta substituted compounds
4f and 4g. This confirmed an unexpected regiosensitivity of the
potential P-gp binding site for the presentation of the function-
ally important groups and thus gives novel insight into the SAR
of mdr modulators. The fact that selected compounds 4a and
4b did not induce P-gp in the inducible cell model compared
to daunorubicin is of favor for the novel P-gp inhibitors with
respect to clinical studies and a great advantage compared to
known inhibitors.
Experimental Section
General. Commercial reagents were used without further
purification. 1H NMR (400 MHz) spectra were run with tetra-
methylsilane as an internal standard. Mass spectra were recorded
with an AMD 402 using EI and a Finnigan-LCQ Classic mass
spectrometer using ESI techniques. Elemental analyses were
performed with a Leco CHNS-932 apparatus. TLC analyses were
carried out with E. Merck 5554 silica gel plates.
3,9-Dibenzyl-6,12-bis(3-benzyloxyphenyl)-1,5,7,11-tetrakishy-
droxymethyl-3,9-diazahexacyclo[6.4.0.02.7.04.11.05.10]dodecane (4f).
The synthesis of the 1,4-dihydropyridines 2a and 2b has been
reported in ref 19.
1
Yield 0.021 g (29%); mp 111-117 °C; H NMR (DMSO-d6) δ
7.63 (d J ) 8.7 Hz, 1 H), 7.45-7.20 (m, 21 H), 7.04 (m, 3 H),
6.96 (s, 1 H), 6.78 (d J ) 10.8 Hz, 1 H), 6.71 (d J ) 10.8 Hz, 1
H), 5.01 (s, 2 H), 4.84 (s, 2 H), 4.44 (m, 4 H), 4.17 (s, 4 H), 3.68
(s, 2 H), 3.15 (m, 8 H), 2.95 (s, 4 H); MS (ESI) m/z ) 850 (M +
Na+). Anal. (C54H54N2O6) C, H, N.
General Procedure for the Formation of Benzylamines 1e,g
from Corresponding Aldehydes.20 The aldehyde (1 equiv, 10 mmol)
and freshly distilled triethylamine (1.1 equiv, 11 mmol) were added
to a cold solution of hydroxylamine hydrochloride (1 equiv, 10
mmol) in anhydrous acetonitrile (50 mL). The mixture was stirred
for 1 h. Then phthalic anhydride (1.05 equiv, 10.5 mmol) was added
in portions under nitrogen atmosphere. The solution was heated
under reflux until nearly no more reaction intermediates were
detectable by TLC. Workup procedure included solution concentra-
tion, stirring with dichlormethane (60 mL), and filtration followed
the washing with 5% ammonia-water for several times, drying
over sodium sulfate, filtration, and evaporation to dryness. The
residual oil was dissolved in petrol ether/ethylacetate mixtures from
which the nitriles crystallized. The crude nitriles (1 equiv, 30 mmol)
were dissolved in anhydrous diethyl ether (30 mL), and the solutions
were added dropwise to a solution of lithium aluminum hydride
(1.2 equiv, 36 mmol) in anhydrous diethyl ether (55 mL) at 4 °C.
After the mixture was stirred at low temperature for 2 h, portions
of water were added. After separation of the organic layer, drying
over sodium sulfate, and filtration, the amines crystallized under
evaporation.
General Procedure for the 1,4-Dihydropyridine Formation of
2c-g.19 Aromatic aldehyde (1 equiv, 10 mmol), ethyl propiolate
(2 equiv, 20 mmol), and benzylamine derivatives (1 equiv, 10
mmol) were dissolved in freshly distilled acetic acid (1 mL). The
mixture was stirred for 1-2 h at 100 °C. After the mixture was
cooled, sufficient water was added (∼10 mL) and then several
extractions followed with chloroform (30 mL). After drying of the
extracts over sodium sulfate and filtration, the solutions were
evaporated and the oily residues were dissolved in ethanol or diethyl
ether from which the compounds 2 crystallized.
3,9-Bis(3-benzyloxybenzyl)-1,5,7,11-tetrahykisdroxymethyl-6,12-
diphenyl-3,9-diazahexacyclo[6.4.0.02.7.04.11.05.10]dodecane (4g).
1
Yield 0.050 g (69%); mp 219-225 °C; H NMR (DMSO-d6) δ
7.81 (d J ) 7.4 Hz, 2 H), 7.40-7.28 (m, 12 H), 7.21 (t J ) 8.0,
7.6 Hz, 2 H), 7.12-7.00 (m, 6 H), 6.95 (s, 2 H), 6.89 (dd J ) 7.6,
2.4 Hz, 2 H), 6.85 (dd J ) 8.0, 2.4 Hz, 2 H), 4.91 (s, 4 H), 4.50
(t J ) 4.4 Hz, 4 H), 4.12 (s, 4 H), 3.65 (s, 2 H), 3.16 (dd, J ) 10.4,
4.4 Hz, 8 H), 2.96 (s, 4 H); MS (ESI) m/z ) 828 (M + H+). Anal.
(C54H54N2O6) C, H, N.
P-gp Inhibiton Assay.15 Cultured cells from mouse T lymphoma
parental cell line L5178 and the P-gp expressing subline L5178Y
were adjusted to a concentration of 2 × 106 cells/mL in serum-
free McCoys 5A medium. Aliquots of 0.5 mL were placed in
Eppendorf tubes. Test compounds were added from stock solutions
in dmso. After incubation for 10 min at rt, rhodamine 123 was
added with a final concentration of 5.2 µM. Incubation continued
for 20 min at 37 °C. After washing twice with phosphate-buffered
saline (PBS), the samples were resuspended and the 1 × 104 cells
were measured by flow cytometry. Fluorescence activity ratios were
calculated by the relation of the determined fluorescence in the
inhibitor treated P-gp expressing subline to the parental cell line
with each fluorescence value corrected to that of the untreated
control.
Lipophilicity Calculations. Molecules were constructed and log
P values were determined with seven calculation programs (ALOGPs,
AC_logP, AB/LogP, COSMOFrag, miLogP, KNOWWIN and
XLOGP).21 The resulting mean values were given by the ALOGPS
2.1 program.21
Induction Studies. Cells of the gastric carcinoma cell line were
cultured as described in six-well plates with a density of 5 × 105
cells/well.22 After addition of the compounds for P-gp induction
General Procedure for the 3,9-Diazatetrasterane 3a-g Forma-
tion. 1,4-Dihydropyridines 2 (1 equiv, 1.2 mmol) were dissolved
in dried THF (30 mL) in a quartz flask. After syringing with argon,
the solution was irrdiated with the light of Ultra-Vitalux lamps (λ
> 270 nm) from a distance of 60 cm at rt. The product formation