Metalloproteinase-2 SelectiVe Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1413
tumor in severe combined immunodeficiency (SCID). In this model,
the tumor cells were transfected with the luciferase gene as a
reporter marker. Tumor growth and tumor dissemination was
followed weekly with a CCCD camera using luciferin as a detecting
device.20 The mice were treated for six weeks by daily intraperi-
toneal injections, except weekends, of 50 mg/kg of the tested drug.
Instrument. HPLC-MS: Waters Millennium HPLC-MS instru-
ment equipped with Micromass ZQ detector, Waters 600 Controller
gradient pump, and Waters 717 autosampler.
Column. Waters Xterra MS C18 column 2.1 × 150 mm was
used with the following settings: nitrogen flow, 500 L/h; desolvation
temp, 400 °C; source temp., 150 °C; cone voltage, 15 V.
Mobile Phase. The mobile phase consisted of 3% methanol, 97%
water, 0.1% formic acid, and 0.05% trifluoroacetic acid.
Flow rate. A flow gradient was applied as follows: 1–3 min,
0.1 mL/min; 3–3.5 min, flow increase to 0.2 mL/min; 3.5–12 min,
0.2 mL/min; 12–13 min, flow rate decrease to 0.1 mL/min; and
13–15 min, 0.1 mL/min. No change in the mobile phase constituent
was done during the analysis.
Pharmacokinetic Experiments. Diffusion in Isolated Rat
Intestine (Ussing). cis-ACCP was dissolved in rat mucosa buffer
to create a solution with the concentration of 600 µg/mL, and 3
mL of this solution was inserted into the mucosal side of Ussing
diffusion chambers containing different intestine segments of the
same rat. Four Wistar rats were sacrificed and intestine segments
of the jejunum, ileum, and column were assembled in the Ussing
chambers to determine the diffusion of cis-ACCP. Samples of 100
µL were withdrawn at time 0 and 150 min from the mucosal side
and at times 30, 60, 90, 120, and 150 min from the serosal side
and stored immediately at -20 °C until the analysis. The withdrawn
amounts of the solutions from the Ussing chambers were replaced
by fresh mucosa buffer.
Acknowledgment. This work was supported in part by the
Ministry of Science of Israel and in part by The German Israeli
Foundation for Scientific Research and Development (GIF) to
E.B. and R.R. and, in part, by the Grass Center for Drug Design
and Synthesis of Novel Therapeutics. A.H., R.R., and E.B. are
affiliated with the David R. Bloom Center of Pharmacy, in the
School of Pharmacy, Faculty of Medicine, The Hebrew Uni-
versity of Jerusalem.
Pharmacokinetic Evaluation of cis-ACCP in Rats and Mice.
Intravenous, Oral, and Intraperitoneal Administration of cis-
ACCP to Rats. cis-ACCP was administered intravenously (50 mg/
kg), orally (150 mg/kg), or intraperitoneally (150 mg/kg) to mice
weighing 320–370 g. The compound was dissolved in normal saline/
water (60/40; solution concentration: 20 mg/mL).
Following intravenous injection, blood samples of 250 µL were
withdrawn at time points 5, 10, 20, 30, 60, 90, 120, 180, and 240
min. Following oral and intraperitoneal administration, blood
sampling was extended to 8 and 24 h, respectively. The blood was
centrifuged at 5000 rpm for 5 min, and 100 µL of the supernatant
plasma was transferred to Eppendorf tubes and stored at -20 °C
until being analyzed. Rats were kept in metabolic cages, and urine
samples were collected 24 and 48 h after the start of each
experiment and stored at -20 °C for the evaluation of urinal
excretion of cis-ACCP.
Supporting Information Available: The organic synthetic
procedures, analytical data for the final compounds and some
intermediates, and graphs of the time dependence and mode of
inhibition by cis-ACCP are presented. This material is available
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Calibration curves have been prepared on the day prior to the
analysis. Samples of plasma containing the following concentrations
of cis-ACCP were prepared by spiking the compound in fresh blank
plasma: 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 75, 100, 200, and 400
µg/mL. To prepare a calibration curve in the urine, concentrations
of 10, 25, 50, 100, 200, 500, and 1000 µg/mL of cis-ACCP in blank
urine were prepared. The samples were then treated as mentioned
above.
(6) It should be mentioned that both ACCP studied are racemic mixtures.
Our attempts to synthesize an enantiomerically pure cis-isomer, so