A. P. Massey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2635–2639
2637
smoothly, the amidoxime was resistant to hydrogenation under
these conditions, resulting in the formation of amidoxime 8. The
formation of 8 was accompanied by further reduction of the
C(6)-chloro substituent, which led to formation of 8-H as the major
product of the reaction. All subsequent attempts to reduce the ami-
doxime moiety of 7 to its amidine (e.g., catalytic hydrogenation,
SnCl2)23 either failed or gave similar mixtures of 8 and 8-H. Simi-
larly, attempts to reduce the amidoxime group of the dichloropyr-
to their benzoylguanidine congeners. The amidoxime analog (8)
demonstrated a significant reduction in potency as a uPA inhibitor
comparable to that of the amidine derivatives. Dechlorination (8-
H) had no impact on altering inhibitory activity. Addition of the
benzyl ester formation at the C5-glycine moiety of the amidoxime
derivative (7) partially restored uPA inhibitory potency (IC50
74 lM) with very slight inhibition of tPA (IC50 200 lM). The enzy-
matic assay was done as described.25,26
azine 9, readily prepared by reaction of nitrile
5
with
Amiloride is cytostatic to several cancer cell types12 and has
been reported to produce apoptotic cell death in several others.13
hydroxylamine, also failed to deliver the corresponding amidine
10. We were gratified to find, however, that reaction of nitrile 5
according to the Garigipati protocol led directly to the C(2)-ami-
dine.24 Treatment of 5 with in situ generated MeAl(Cl)NH2 gave
amidine 10 in good yield without complications arising from sub-
stitution of the usually reactive C(5)-chloro group. The robust nat-
ure of the C(5)-chloro became apparent in the next step as we
attempted to substitute it with glycine as in our previous exam-
ples. Reaction of amidine 10 with benzyl glycine under the stan-
dard conditions gave adduct 11 in 17% yield. Attempts to
improve the substitution by subjecting the reactants to more forc-
ing conditions led to lower isolated yields of 11. Hydrogenation of
11 (H2, 10% Pd/C, EtOH) gave a 2.3:1 mixture of the debenzylated
products 2.2-H in 81% yield. The amidines 2 and 2-H were readily
separated using C18 reverse phase chromatography (CH3CN: 0.1%
aq. TFA gradient).
The availability of dichloropyrazine amide 4 led us to prepare
the corresponding benzyl glycine conjugate to serve as a neutral
C(2)-analog for comparison to the more basic C(2)-amidoxime
and C(2)-amidine analogs. In contrast to the reaction of 10, nucle-
ophilic aromatic substitution on 4 (Scheme 3) proceeded smoothly
to afford conjugate 12. This ease of this transformation mirrors clo-
sely our previously reported synthesis of the benzyl glycine conju-
gate of amiloride (e.g., 13?14).5,6
Previously, our group reported that amiloride (ꢁ200
lM) causes
caspase-independent, non-apoptotic cell death of human malig-
nant glioma cells.14,15 Despite its low potency as a selective anti-
cancer agent, amiloride’s non-toxicity in humans makes it appeal-
ing to explore as a potential candidate for preventing recurrence of
highly invasive and metastatic cancers.16 The lactate dehydroge-
nase (LDH) assay quantifies changes in cytosolic LDH released from
dying and dead cells and distinguishes drug-induced anti-glioma
cytotoxicity from cytostatic effects, unlike the more commonly re-
ported metabolism-based, tetrazolium live cell assays. Using the
LDH assay,27 amiloride (250
lM) kills 50% of U87 glioma cells at
24 h (Table 1). Benzylglycinyl substitition at the C5 position of
amiloride (14) or its amidine homolog (11) confer respective uPA
inhibitor potencies of IC50 = 3 and 4 lM, but 14 is more cytotoxic
than 11. The corresponding glycinyl acids at the C5 position (1
and 2) do not demonstrate glioma cytotoxicity, and 2 lost most
of its uPA inhibitory activity. Substitution of the amidoxime group
at C5 eliminated uPA inhibitory and anti-glioma cytotoxic
potencies.
Amiloride, an acylguanidine, is one of the most selective inhib-
itors of uPA, relative to other proteases. Our data is in agreement
with studies showing that amiloride has an IC50 reported to be be-
tween 7 and 50 l
M for uPA with IC50 >1000 for tPA or plasmin.17
In Table 1, the addition of a glycine moiety to amiloride to form
(1) minimally reduced the inhibitory potency of the free acid for
uPA (IC50 17 lM), as compared with the amiloride (IC50 7 lM),
while preserving selectivity as a protease inhibitor (Table 1). The
absence of the C6 chloro group (1-H) reduced inhibitory potency
Compared with other trypsin-like serine proteases, the S2 and
S3/S4 pockets of uPA are reduced in size because of the 99-inser-
tion loop.18 Nienaber and colleagues utilized the crystalline struc-
ture of re-engineered urokinase to demonstrate that amiloride
utilized its C(6) chloride to occupy an auxillary structural subsite
adjacent to the primary binding pocket and which conferred the
inhibitory specificity observed with amiloride.11 The unique bind-
ing mode of this class of uPA inhibitors utilizes interactions at the
S1’/S2’ subsites, which enhance their inhibitory selectivity and
potency.19,20 Our selection of amiloride as the initial intracellular
uPA inhibitor is based upon its high selectivity for uPA inhibition
and its minimal toxicity profile in humans.
against uPA (IC50 47
lM), but did not alter its selectivity as an
uPA inhibitor. Benzylester formation (14) enhanced inhibitory
potency (IC50
inhibitor.
3 lM), while preserving selectivity as a protease
Amidine substitution for the C(5) benzoylguanidine group of
the free acid form of C2-glycinyl amiloride (2) markedly reduced
its potency as a uPA inhibitor (IC50 237 lM). The absence of the
C6 chloro group (2-H) did not alter the inhibitory potency of the
amide glycinyl derivatives, relative to uPA or alter its selectivity
against the other proteases. By contrast, formation of the benzyl
ester at the C2 substituted glycine moiety (11) restored inhibitory
potency (IC50 4 lM) to that of amiloride and of 14 while not alter-
ing selectivity as a protease inhibitor (Table 1).
C2-amidine derivatives of amiloride would appear to be struc-
turally analogous to the acylguanidine inhibitors of uPA (Fig. 1).
A significant increase in chemical stability and potency as a uPA
inhibitor has been reported by incorporating amidine moities,
but with a proportional gain in inhibitory potencies of tPA, trypsin,
and thrombin.7 However, it has been proposed that selectivity of
C2-amidine derivatives for uPA should be maintained by the intro-
duction of a halogen at the C6 position.4 While maintaining the
pyrazine core of amiloride we demonstrated that de-chlorination
significantly reduced uPA inhibitory potencies of the C2-amidine
and C2- amidoxime derivatives. Furthermore, no gain in inhibitory
potencies was noted with other related proteases. Inclusion of the
benzyl ester on the 2R glycinyl group significantly improved inhib-
itory potency of uPA while maintaining selectivity towards other
proteases. This suggests that hydrophobic stabilization by the 2R
group contributes towards uPA inhibitory activity of these pyra-
zine-based derivatives and agrees with independent data obtained
using amidine aryl cores.3,8
The C2 amidoxime glycinyl amiloride derivatives explored the
impact of the more basic amidoxime and amide inhibitors, relative
Cl
Cl
N
N
W
Z
N
N
W
a
5
NH
RO C
N
H
NH
2
2
2
4: W = C(O)NH
2
12: R = Bn, Z = Cl, W = C(O)NH
2
13: W = C(O)N=C(NH )
2 2
14: R = Bn, Z = Cl, W = C(O)N=C(NH )
2 2
2 2
2 2
b
1: R = H, Z = Cl, W = C(O)N=C(NH )
1-H: R = H, Z = H, W = C(O)N=C(NH )
Amiloride has been reported to kill human glioma cells and
other cancer cells, including malignant breast cancer cell lines,
in micromolar concentrations.14 Here we demonstrate that
Scheme 3. Additional C(2)-analogs. a12: BnO2CCH2NH3 TsOÀ, Et3N, DMF, 60 °C,
18 h, 45%; bH2, cat. 10% Pd/C, EtOH, 48 h, 83% (1.1-H, 1:1.5).
+