X. Zhang et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127939
exploration on the left-side terminal R group of structure Ia in Table 1.
Generally, an unmodified guanidine group was essential for both high
EP and trypsin inhibitory potency. Methylation of the guanidine group
resulted in a drop in EP and trypsin inhibition at 0.1 μM, as illustrated by
compounds 9 and 10 with <50% inhibitory activity (40.55% and
15.32% for EP, 31.39% and 9.6% for trypsin, respectively) compared to
compound 7 with 97.76% inhibition for EP and 97.16% inhibition for
trypsin. Extending the guanidine group by one carbon dramatically
Fig. 1. Design of novel potent and gut-restricted EP inhibitors.
reduced EP and trypsin inhibition at 1 μM from 97.75% and 100.28% for
The syntheses of representative compounds, triazolopyridine 7 and
imidazopyridine 8 are shown in Scheme 1. 6-Bromo-2-nitropyridin-3-ol
1 was protected with BnBr and K2CO3 in DMF followed by reduction
with Fe and NH4Cl to afford the corresponding aminopyridine 2 in
moderate yield. Formation of triazolopyridine 3 was achieved by
condensation of aminopyridine 2 with DMF-DMA and hydroxylamine
followed by TFAA promoted cyclization in 76% yield for 2 steps. Negishi
coupling of bromide 3 with commercially available (2-tert-butoxy-2-
oxoethyl)zinc(II) bromide in the presence of Pd2(dba)3 as the catalyst
and XPhos as the ligand at 110 ◦C under microwave irradiation followed
by debenzylation by hydrogenolysis afforded the corresponding phenyl
t-butyl acetate 4. Coupling of phenol 4 with 4-(2,3-bis(tert-butoxy-
carbonyl) guanidino)benzoic acid in the presence of EDCI and DMAP
(cat) in DCM at room temperature followed by acidic deprotection with
TFA gave triazolopyridine 7 in good yield. Coupling of aminopyridine 2
with 2-chloro-acetaldehyde in EtOH at 80 ◦C gave imidazopyridine 5 in
57% yield. An analogous synthetic route involving Negishi reaction,
debenzylation, EDCI-mediated coupling and TFA deprotection afforded
the corresponding imidazopyridine 8. Syntheses of all other compounds
are described in detail in the supporting information.
7 to 43.67% and 19.88% for 11. Cyclization of the guanidine group to
produce 1-phenylimidazolidin-2-imine 12 maintained moderate to good
EP and trypsin inhibitory activity at 1 and 10 μM, however, cyclization
or di-methylation of the two terminal nitrogen atoms of the guanidine
group essentially abolished the inhibition of EP and trypsin, as shown by
N-phenyl-4,5-dihydro-1H-imidazol-2-amine 13, N-phenyl-1,4,5,6-tetra-
hydropyrimidin-2-amine 14 and dimethylated guanidine 15 with <15%
inhibition at 10 μM. These results suggest that steric factors may play a
dominant role in ligand-EP and ligand-trypsin binding efficiency. In
contrast, replacement of the unsubstituted guanidine with its bio-
isostere, acetimidamide (16), reduced percent EP inhibition at 0.1 μM
but maintained EP inhibitory activity at 1 and 10
μ
M relative to com-
pound 7. It was noted that 16 was significantly less potent at 1
μ
M with
67.99% trypsin inhibition compared with 7. Reducing the basicity of the
acetimidamide by substitution with a hydroxy group (17) on the nitro-
gen abolished both EP and trypsin inhibitory activity. Benzimidamide
18, a truncated bioisostere of the guanidine group, displayed reduced
percent inhibition at all concentrations, indicating the importance of the
size and length of the terminal basic group for EP and trypsin inhibitory
activity. Addition of a nitrogen atom to the imidamide group did not
yield improved EP or trypsin inhibitory activity as shown by imidohy-
drazide 19. Other efforts to modify the imidamide group by reducing
basicity (20 and 21) or cyclization (22) all failed to boost inhibitory
activity against EP and trypsin. Overall, EP inhibitory activity emerged
in parallel to trypsin inhibitory activity in this chemical scaffold. The
results of the SAR study of the structure Ia on the R group made it
apparent that the guanidine group binds to a narrow and highly selective
site of the EP binding pocket, which was recently confirmed by an X-ray
crystallographic study of a camostat analogue containing the guanidine
benzoate structure.7 In the fragment/EP complex, the carbox-
ymethylbenzylguanidine moiety (fragment) occupied the S1 subsite,
forming a very tight salt bridge with the sidechain of Asp181 at the base
of this pocket.
Compounds were screened in the biochemical assay with recombi-
nant full-length human EP expressed in CHO cells. Progress curves of
protease activity were measured by monitoring the fluorescence in-
tensity of the cleavage product 2-naphthylamine (NA) from the peptide
substrate, GD4K-NA, at excitation and emission wavelengths of 340 and
410 nm, respectively.17 Bovine trypsin was purchased from Sigma-
Aldrich (Cat No. T1426), and 7-amino-4-methylcoumarin (AMC) con-
jugated peptide H-D-cyclohexylalanine (D-CHA)-AR-AMC was used as
the substrate in the trypsin inhibition assay. The protease activity of
trypsin was followed by measuring AMC production by the emission
fluorescence intensity of AMC at 460 nm with the excitation at 375 nm,
in the same assay buffer as used in the EP assay.18 To accelerate the
primary screening process, compounds were first screened for EP and
trypsin inhibition at concentrations of 0.1, 1 and 10
pounds with > 50% inhibition at 0.1 M were advanced into full kinetic
studies of concentration-dependent inhibition. We began our SAR
μM. Selected com-
We then shifted our efforts to establishing the SAR of the left-side H
ring (Table 2). The 1,4-relationship of the guanidine benzoate core is
essential for the inhibition of EP and trypsin, as demonstrated by the loss
μ
Scheme 1. Reagents and conditions: a) BnBr, K2CO3, DMF at 70 ◦C (80%); b) Fe, NH4Cl in EtOH at 80 ◦C (85%); c) DMF-DMA, NH2OH.HCl, NaHCO3 in IPA 55 ◦C
(82%); d) TFAA in THF 0 ◦C to r.t. (93%); e) 2-chloro-acetaldehyde in EtOH at 80 ◦C (57%); f) (2-tert-butoxy-2-oxoethyl)zinc(II) bromide, Pd2(dba)3, XPhos in THF
110 ◦C under MW (67–98%); g) H2 (1 atm), 5% Pd/C in EtOAc (64–69%); h) 4-(2,3-bis(tert-butoxy-carbonyl) guanidino)benzoic acid, EDCI, DMAP in DCM at r.t.
(61–71%); i) TFA in DCM, r.t. (52–88%).
2