CGS 27023A, a Stromelysin Inhibitor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2531
The organic layer is dried (Na2SO4), and the solvent is
evaporated. The product is purified by silica gel chromatog-
raphy (first 50% ethyl acetate, then 5% methanol/methylene
chloride) to give diethyl [2-(4-morpholino)ethyl]acetamidoma-
lonate (8.0 g, 45% yield): NMR (250 MHz, CDCl3) ∂ 7.1 (s, 1
H), 4.2 (m, 4 H), 3.6 (m, 4 H), 2.35 (m, 8 H), 2.0 (s, 3 H), 1.3
(m, 6 H).
of Thonar.30 Sulfated glycosaminoglycans are measured by
first digesting the synovial lavage with streptomyces hyalu-
ronidase and then measuring DMB dye binding using the
method of Goldberg.31
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Eth yl 2-Aceta m id o-2-[2-(4-m or p h olin o)eth yl]a ceta te.
Diethyl [2-(4-morpholino)ethyl]acetamidomalonate (8.0 g, 25.6
mmol) is dissolved in ethanol (128.0 mL). Sodium hydroxide
(4.55 mL of a 6 N aqueous solution, 27.35 mmol) is added, and
the reaction mixture is stirred at room temperature for 24 h.
The ethanol is then evaporated, the residue is diluted in water
and washed several times with ether, and then the aqueous
phase is acidified with concentrated hydrochloric acid to pH
) ∼5. The solution is evaporated to dryness, suspended in
toluene (300.0 mL), and refluxed for 3 h. After being cooled
to room temperature, the reaction mixture is diluted with
chloroform (300.0 mL) and the mixture is filtered through
Celite. The filtrate is evaporated to give ethyl 2-(acetamido)-
2-[2-(4-morpholino)ethyl]acetate (6.0 g, 91% yield): NMR (300
MHz, CDCl3) ∂ 7.5 (d, 1 H), 4.6 (m, 1 H), 4.2 (m, 2 H), 3.7 (m,
4 H), 2.5 (m, 6 H), 2.0 (s, 3 H), 1.9 (m, 2 H), 1.3 (t, 3 H).
2-Am in o-2-[2-(4-m or p h olin o)eth yl]a cetic Acid Dih y-
d r och lor id e. Ethyl 2-acetamido-2-[2-(4-morpholino)ethyl]-
acetate (4.2 g, 16.28 mmol) is dissolved in 6 N hydrochloric
acid (100.0 mL), and the reaction mixture is refluxed for 4.5
h. The water is then evaporated, and the product is azeotroped
dry using toluene to give 2-amino-2-[2-(4-morpholino)ethyl]-
acetic acid dihydrochloride (4.0 g, 94% yield): NMR (250 MHz,
D2O) ∂ 4.1 (m, 3 H), 3.85 (t, 2 H), 3.6 (t, 2 H), 3.45 (m, 2 H),
3.2 (m, 2 H), 2.4 (m, 2 H).
This amino acid is then carried through the general scheme
as described above to provide 68.
Biologica l Assa ys. Stromelysin inhibitory activity is based
on the hydrolysis of substance P using a modified procedure
of Harrison.27 In this assay, substance P is hydrolyzed by
recombinant human stromelysin to generate a fragment,
Substance P 7-11, which can be quantitated by HPLC. In a
typical assay, a 10 mM stock solution of a compound to be
tested is diluted in the assay buffer to 50 M, mixed 1:1 with
8
g of recombinant human stromelysin (mol wt 45-47 kDa,
2 units, where 1 unit produces 20 nmol of substance P 7-11
in 30 min) and incubated along with 0.5 mM substance P in a
final volume of 0.125 mL for 30 min at 37 °C. The reaction is
stopped by adding 10 mM EDTA, and substance P 7-11 is
quantified on RP-8 HPLC. The IC50 is calculated from control
reaction without the inhibitor. The mean Ki values reported
in the tables have been derived from the mean IC50 according
to the method of Waley.28 The mean IC50 was obtained from
two independent measurements, and the standard deviation
of the mean was less than 10%. To help validate this assay,
the best Merck lead was made and found to have a Ki of 361
nM (versus a reported Ki ) 470 nM).29
Ex vivo pharmacokinetic studies were usually carried out
in rats. Compounds were dosed in DMSO/cornstarch at 75
mol/kg po, after which the rats were bled into heparinized
tubes at various time points (typically 1, 2, and 3 h later). After
centrifugation at 2000 rpm, the plasma was removed, and the
drug was extracted with acetonitrile. The drug level was then
determined by incubating this sample with recombinant
human stromelysin and substrate and comparing the inhibi-
tion observed with a standard curve. It should be emphasized
that this method does not directly quantitate the amount of
parent compound.
The efficacy of compounds in vivo is determined by studying
them in rabbits. Typically, four rabbits are dosed orally with
a compound at a dose of 75 mol/kg in 5 mL of fortified corn
starch per kilogram of body weight, up to 8 h before intra-
articular injection in both knees (N ) 8) with 40 units of
recombinant human stromelysin (dissolved in 20 mM Tris, 10
mM CaCl2, and 0.15 M NaCl at pH 7.5). Two hours later the
rabbits are sacrificed, synovial lavage is collected, and keratan
sulfate (KS) and sulfated glycosaminoglycan (S-GAG) frag-
ments released into the joint fluid are quantitated. Keratan
sulfate is measured by an inhibition ELISA using the method