B.J. Buckley, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 2. X-ray co-crystal structures of A) 7 (PDB 6AG2;
1.8 Å), B) 8 (PDB 6AG3; 2.5 Å), C) 13 (PDB 6AG7;
1.9 Å) and D) 15 (PDB 6AG9; 1.6 Å) bound to the
active site of uPA. A sulfate of crystallization is
shown, where detected. X-ray statistics are provided
in the Supplementary Material.
fold higher activity (15 Ki = 302 nM). The potency increases achieved
with this series generally tracked with the trends seen with the corre-
sponding 5-hexamethyleneamiloride derivatives we recently re-
ported.18 An exception was seen with the 2,4-dimethoxy analog 8,
which failed to recapitulate the > 55-fold potency increase seen with
its corresponding HMA analog.18
potency
The gains in uPA potency (relative to amiloride) achieved with the
6-substituted amiloride series herein were relatively modest compared
creases exceeding 110-fold were observed.18 The best inhibitor in the
current series, 4-methoxypyrimidine 7, showed only a 12-fold im-
provement relative to amiloride. This trend can be explained in part by
the decreased interactions seen between the 6-substitutents and the S1β
subsite arising from their coplanarity with the pyrazine core. Additional
van Der Waals interactions contributed by the azepane ring interacting
with the S4 pocket of uPA presumably also play a role.
The improved potency of 15 led to exploration of a small series of 5
and 6-substituted 2-benzofuranyl derivatives 16–22 to understand the
effects of substituents around the benzofuran. However, none of the
substituted benzofurans showed improved potency relative to 15. 5-Br
substitution 19 was particularly unfavorable, causing a 1.5-fold de-
crease in activity relative to amiloride (~12-fold loss relative to 15).
Altering the connectivity of the benzofuran to the pyrazine (i.e. 5-
benzofuranyl 23) caused only a small decrease in potency relative to 15
and a slight increase was observed with the 2,3-dihydro-5-benzofuranyl
24 relative to amiloride.
To explore uPA selectivity within the series, compound 15 was
screened against a panel of closely related TLSPs using chromogenic
enzyme inhibition assays (Table 2). A high degree of uPA selectivity
TLSPs tested. A similar degree of selectivity was observed with
amiloride, as previously noted.18
The binding pose of the pyrazine acylguanidine unit in the S1
pocket of uPA was not significantly altered from that of amiloride in
most cases by addition of the 6-substituents, as evidenced by X-ray co-
crystal structures of 7, 13 and 15 bound to uPA (Figs. 2,3 and Fig. S1).
subtle shifting of the pyrazine core out of the S1 pocket lengthened the
key salt bridge interaction between the guanidine and Asp189 relative
to other derivatives (and 1), thus contributing to its reduced potency.
Another common feature observed with 13 and 15 (and to a lesser
extent 7) was an almost coplanar positioning of the pyrazine core with
the 6-(het)aryl substituents, which appeared to limit interactions with
the S1β subsite. This orientation differed from the corresponding HMA
analogs, where larger dihedral angles about the pyrazine bond axis
were seen, which appeared to confer better occupancy of S1β by the
respective 6-(het)aryl groups.18 An exception to this was observed with
compound 8, whose dimethoxypyrimidine had rotated 72˚ to project
the 2-methoxy group away from S1β and towards bulk solvent. This
was in contrast to its matched HMA analog, where a similar degree of
rotation in the opposite direction allowed partial occupancy of S1β by
the corresponding 2-methoxy group, leading to a considerable boost in
To further profile target selectivity, amiloride 1 and derivatives 7
and 15 were screened against 85 serine hydrolases using a high-
throughput EnPlex assay (Fig. 3).27 Amiloride 1 showed slight inhibi-
LACTB), consistent with previous reports6 and solution phase enzyme
assay data (Table 2). High uPA selectivity was similarly observed for
both 7 and 15, with trypsin and prolylcarboxypeptidase (PRCP) being
the only enzymes showing slight inhibition across the serine hydrolase
superfamily.
Species selectivity between human and mouse uPA (MuPA) is a
known issue for the development of reversible, active-site inhibitors of
human uPA (HuPA). This issue is particularly apparent for inhibitors
that target the S1β subsite, where the improved potencies typically seen
against the human enzyme are not recapitulated with MuPA.28 This
trend was observed for the 2-benzofuranyl derivative 15, which showed
33-fold lower activity against the murine variant (MuPA
IC50 = 14.2 μM vs HuPA IC50 = 0.43 μM). In contrast, amiloride with
its smaller 6-chloro substituent showed equivalent potency against
3