1316
Vol. 53, No. 10
CYP3A4 inhibition. It was thus tempting to speculate that sis [Symmetry® C18, 4.6 mmfꢃ100 mm, water–MeOH–THF (66 : 24 : 10) at
1 ml/min]. The time dependency of 6b-hydroxytestosterone concentrations
was measured with and without test compounds. The IC50 value was calcu-
lated by linear interpolation. The initial-rate constant for inactivation (k,
minꢁ1) was estimated from the slope of the log-linear regression line of
residual CYP3A4 activity vs. the preincubation time profile. Ki and kinact
were determined using Eq. 1, where I is the test compound concentration,
kinact is the maximum rate constant inactivation, and Ki is the test compound
concentration that produces half the maximal rate of inactivation.
the N-benzyl moiety of our leads could be the structural fea-
ture responsible for irreversible CYP3A4 inhibition. There-
fore we focused our optimization strategy on modifying this
molecular fragment.
Whereas the introduction of 2-F (14) only showed mar-
ginal improvement, the corresponding 2-Cl derivative (15)
showed much better properties, inhibiting CYP3A4 only
weakly after 30 min of preincubation (IC50ꢀ24 mM). This ef-
fect was confirmed after introduction of an additional sub-
stituent in 4-position (18, IC50ꢀ27 mM after 30 min). The 3-
and 4-chloro-derivatives 16 and 17 however, were still potent
time-dependent CYP3A4 inhibitors, suggesting the impor-
k
inactꢃI
(1)
k ꢀ
KiꢄI
1-(2-Chlorobenzyl)-2-[2-(3-tert-butyl-ureido)-ethyl]-1H-benzimida-
zole-5-(3,4-difluorobenzyl)sulfonamide (19) A solution of N-(tert-butyl)-
Nꢅ-(2-{1-[(2-chlorophenyl)amino]-5-amino-1H-benzimidazol-2-
tance of a large substituent in ortho position. Indeed, keeping yl}ethyl)urea (7: XꢀCl, 1.2 g, 2.9 mmol) was suspended in conc. HCl/HOAc
and converted to a diazonium salt by adding a solution of NaNO2/water
(216 mg, 3.14 mmol) portion wise (1 h) at 0 °C. In a different flask, CuCl2
(291 mg, 1.71 mmol) was added to SO2-saturated acetic acid and stirred
under continous bubbling of SO2 for 20 min. The solution of the diazonium
the 2-Cl substituent in the molecular structure led to the
identification of compounds 19—21 with IC50 of 10—27 nM
and no CYP3A4 inhibition activity up to 30 mM (19, 21).12)
Confirming this hypothesis, the incorporation of steric bias in
the adjacent methylene bridge also provided an LHRH in-
hibitor free from CYP3A4 interaction (22).
salt was added to the CuCl2–SO2 mixture at 0 °C and stirred for 30 min.
After further stirring at room temperature for 30 min, the mixture was
quenched with 10 ml of ice water. The whole mixture was extracted 3 times
with EtOAc, and the organic layer was washed with water, saturated
NaHCO3 aq. and brine. The organic solvent was removed in vacuo to give
the crude sulfonyl chloride 8 as a brownish precipitate (81%). LC-MS m/z:
483 (Mꢄ), 421, 384.
The sulfonylchloride 8 (40 mg, 0.07 mmol) was reacted with 3,4-difluo-
robenzylamine (12.3 mg, 0.09 mmol) in CH2Cl2 with triethylamine for 2 h at
room temperature. After removing the solvent, the residue was purified
through preparative thin layer chromatography (CH2Cl2/MeOHꢀ12/1) to
In summary, we have discovered a novel class of benzimi-
dazole LHRH receptor antagonists 19—22, virtually free of
time-dependent CYP3A4 inhibition. We have devised struc-
ture activity relationships indicating the N-benzyl group to be
involved in this unwanted side effect. The structural conclu-
sions drawn for avoiding irreversible CYP3A4 inhibition
might be of value for the optimization of similar structural
frameworks.
give the desired 1-(2-chlorobenzyl-2-[2-(3-tert-butyl-ureido)-ethyl]-1H-benz
-
imidazole-5-(3,4-difluorobenzyl)sulfonamide 19 (32 mg, 73% yield) as a
white powder. 1H-NMR (300 MHz, CDCl3) d: 1.62 (9H, s), 2.96 (2H, t,
Jꢀ5.8 Hz), 3.71 (2H, q, Jꢀ5.8 Hz), 4.12 (2H, d, Jꢀ6.3 Hz), 4.58 (1H, s),
5.32 (1H, t, Jꢀ6.0 Hz), 5.49 (2H, s), 6.50 (2H, m), 7.00 (2H, m), 7.15 (1H, t,
Jꢀ7.6 Hz), 7.27 (3H, m), 7.46 (1H, dd, Jꢀ7.9, 1.2 Hz), 7.72 (1H, dd, Jꢀ8.5,
1.7 Hz), 8.18 (1H, d, Jꢀ1.4 Hz). LC-MS m/z: 590 (Mꢄ), 517, 491. HR-MS
m/z: 590.1821 (Mꢄ) (Calcd for C28H30ClF2N5O3S: 590.1799). mp 157—
158 °C.
1-Benzyl-2-[2-(3-iso-propyl-ureido)-ethyl]-1H-benzimidazole-5-(4-flu-
orobenzyl)sulfonamide (10) 1H-NMR (500 MHz, DMSO) d: 0.98 (3H, s),
0.99 (3H, s), 2.98 (2H, t, Jꢀ6.6 Hz), 3.48 (2H, q, Jꢀ6.6 Hz), 3.63 (1H, q,
Jꢀ6.6 Hz), 3.93 (2H, s), 5.56 (2H, s), 5.90 (2H, m), 7.11 (4H, m), 7.29 (4H,
m), 7.64 (2H, dd, Jꢀ24.0, 8.5 Hz), 8.02 (1H, s), 8.06 (1H, br s). LC-MS m/z:
524 (Mꢄ), 465, 196. HR-MS m/z: 524.2123 (Mꢄ) (Calcd for C27H30FN5O3S:
524.2127). mp 124—125 °C.
Experimental
General Methods Melting points (mp) were determined using a
BÜCHI B-545 melting point apparatus and were uncorrected. The 1H-NMR
1
spectra were recorded on a Bruker DRX-300 (300 MHz for H) spectrome-
ter, Bruker 500 UltraShieledTM (500 MHz for 1H) spectrometer in CDCl3, di-
methyl sulfoxide-d6 (DMSO-d6) or CD3OD using tetramethylsilane (TMS)
as an internal standard. High-resolution mass spectra (HR-MS) were ob-
tained using a Finnigan MAT 95. Liquid Chromatography-Mass spec-
troscopy (LC-MS) was measured using the Micromass Platform LC with a
Shimadzu Phenomenex ODS column (4.6 mmfꢃ30 mm), flushing a mixture
of acetonitrile–water (9 : 1 to 1 : 9) at 1 ml/min flow rate. High performance
liquid chromatographies (HPLC) were conducted by using Shimadzu LC-
10A.
Measurement of LHRH Antagonistic Activity Intracelluar Ca mobi-
lization data were obtained using recombinant CHO cells transfected with
human LHRH receptor cDNA. The cells were pre-incubated with variable
concentrations of test compounds for 20 min at 25 °C. Fluorescence changes
indicating mobilization of cytoplasmic calcium (Flu-3 AM, Molecular
Probes) were measured on a FDSS-3000 machine (Hamamatsu photonics)
after stimulation with 10 nM LHRH. The inhibitory effect of the compounds
was calculated by comparing the integral ratio of actual and intial fluores-
cence.
Metabolite Identification Study The study was carried out with 1-ben-
zyl-2-ethoxy-1H-benzimidazole-5-sulfonamide 9 as a typical structural rep-
resentative of our series. One hour after microsomal incubation the reaction
was quenched with ether and the organic layer was analyzed by HPLC
[Symmetry® C18, 4.6 mmfꢃ100 mm; acetonitrile–aqueous ammonium ac-
etate (pHꢀ6.8) 3 : 7 to 8 : 2 at 1 ml/min] showing two non-base line sepa-
rated peaks [P-1 (72%); tRꢀ5.35 min, m/z (ꢄ1)ꢀ350.4, P-2 (17%);
tRꢀ0.92 min, m/z (ꢄ1)ꢀ242.3], indicating a mixture of debenzylated and 4-
F debenzylated metabolites with remaining starting material 9ꢅ (11%).
Measurement of IC50 against CYP3A4 The incubation mixture [total
amount: 0.2 ml, 0.2 mg/ml of microsomes, 1 mM EDTA, NADPH-generating
system (25 mM glucose-6-phosphate, 2.5 mM NADPꢄ, and 2 units of glu-
cose-6-phosphate dehydrogenase in 100 mM potassium phosphate buffer)],
was pre-incubated with 0.1, 0.3, 1.0, 3.0, 10 and 30 mM of test compounds at
37 °C for 0 and 30 min. The CYP3A4 activity was assayed after initiation
with testosterone (250 mM). The reaction was quenched with EtOAc, the or-
ganic layer was evaporated, and the residues were subjected to HPLC analy-
1-Benzyl-2-[2-(2-fluoro-phenyl)-ethyl]-1H-benzimidazole-5-(4-fluo-
robenzyl)sulfonamide (11) 1H-NMR (300 MHz, CDCl3) d: 1.57 (9H, s),
3.12 (2H, t, Jꢀ6.1 Hz), 3.98 (4H, m), 5.08 (1H, t, Jꢀ6.5 Hz), 5.43 (2H, s),
6.84 (4H, m), 7.03 (4H, m), 7.31 (4H, m), 7.55 (2H, m), 7.81 (1H, d,
Jꢀ1.5 Hz), 8.11 (1H, t, Jꢀ8.5 Hz). LC-MS m/z: 575 (Mꢄ), 465, 439. HR-
MS m/z: 576.1881 (Mꢄ) (Calcd for C30H27F2N5O3S: 576.1876). mp 152—
153 °C.
1-Benzyl-2-[2-(3-tert-butyl-ureido)-ethyl]-1H-benzimidazole-5-(3,4-di-
fluorobenzyl)sulfonamide (12) 1H-NMR (300 MHz, CDCl3) d: 1.20 (9H,
s), 2.99 (2H, br s), 3.70 (2H, q, Jꢀ5.7 Hz), 4.09 (2H, d, Jꢀ6.0 Hz), 4.72 (1H,
s), 5.40 (3H, m), 7.00 (5H, m), 7.33 (4H, m), 7.71 (1H, dd, Jꢀ8.5, 1.6 Hz),
8.12 (1H, d, Jꢀ1.4 Hz). LC-MS m/z: 555 (Mꢄ), 483, 457. HR-MS m/z:
555.7147 (Mꢄ) (Calcd for C28H31F2N5O3S: 555.7153).
1-Benzyl-2-[2-(3-tert-butyl-ureido)-ethyl]-1H-benzimidazole-5-(4-
chlorobenzyl)sulfonamide (13) 1H-NMR (300 MHz, CDCl3) d: 1.23 (9H,
s), 2.95 (2H, t, Jꢀ5.9 Hz), 3.69 (2H, q, 5.8 Hz), 4.12 (2H, d, Jꢀ6.2 Hz), 4.59
(1H, s), 5.33 (1H, t, Jꢀ5.9 Hz), 5.40 (2H, s), 6.37 (1H, br s), 7.03 (2H, m),
7.16 (4H, q, Jꢀ5.3 Hz), 7.35 (4H, m), 7.72 (1H, dd, Jꢀ8.5, 1.6 Hz), 8.19
(1H, d, 1.5 Hz). LC-MS m/z: 554 (Mꢄ), 481, 455. HR-MS m/z: 554.1967
(Mꢄ) (Calcd for C28H32ClN5O3S: 554.1988).
1-(2-Fluorobenzyl)-2-[2-(3-tert-butyl-ureido)-ethyl]-1H-benzimida-
zole-5-(4-fluorobenzyl)sulfonamide (14) 1H-NMR (300 MHz, MeOD) d:
1.24 (9H, s), 3.13 (2H, t, Jꢀ6.4 Hz), 3.60 (2H, t, Jꢀ6.5 Hz), 4.02 (2H, s),
5.61 (2H, s), 6.61 (4H, s), 6.87 (2H, m), 6.97 (1H, t, Jꢀ7.6 Hz), 7.15 (4H,
m), 7.36 (1H, m), 7.50 (1H, d, Jꢀ8.7 Hz), 7.67 (1H, dd, Jꢀ8.6, 1.7 Hz), 8.08
(1H, d, J=1.3 Hz). LC-MS m/z: 556 (Mꢄ), 483, 475.