2
R. J. Neitz et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
to Ser61 while reducing overall lipophilicity (Fig. 1). We were
aided in this effort by inspection of the previously disclosed
X-ray crystal structure of 4 bound to cruzain (pdb 3IUT).13 This
structure reveals that the hydrogen bond to Ser61 in S3 is largely
solvent exposed, implying that more hydrophilic heterocycles at
P3 might retain this interaction while affording the desired reduc-
tion in overall lipophilicity. The S2 sub-site of cruzain is the most
lipophilic and solvent inaccessible and was therefore expected to
contribute more than any other sub-site to a favorable binding free
energy. Accordingly, we considered that a larger, more lipophilic
group at this position might be beneficial in terms of potency.
Among several larger P2 groups explored previously,13 we selected
cyclopentyl and isopropyl for the current study. Finally, the S1 sub-
site in cruzain is rather more solvent exposed and forms relatively
few hydrophobic interactions with the n-Bu side chain of 4 (e.g., a
P1 ethyl analog is nearly as potent as 4). We briefly explored small
gem-dialkyl substitution at P1 (e.g., cyclopropyl) but found such
analogs were devoid of either biochemical or antitrypanosomal
activities yet were more potent inhibitors of key CYPs, possibly a
consequence of a more exposed triazole ring in such analogs. Our
efforts at P1 were thus directed at side chains that retain the larger
cone angle of n-butyl (as in 4) but with the introduction of heteroa-
toms to modulate overall lipophilicity (Fig. 2).
O
O
O
O
H
N
S
S
N
N
H
N
O
Ph
R
1 (K777) R = Me
2 R = propargyl
O
H
N
O
O
Cl
3
N
N
N
O
F
NH
O
F
F
N
F
4
P3
P2
P1
P1'
Figure 1. Structure of the prototypical peptidic cruzain inhibitor 1 (K777), the
related activity-based probe 2, and non-peptidic cruzain inhibitors 3 and 4. The P10–
P3 side chains are indicated.
Glu208. Compound 4 was effective against T. cruzi parasites in two
different cell-based assays and when administered to T cruzi
infected mice at 20 mg/kg BID (ip) for 27 days, compound 4
afforded a haematological cure in 2/4 treated animals.13
New analogs were synthesized using the general synthetic
approaches described previously for 4 and similar analogs.13,14
Briefly, amino acid starting materials bearing the desired P1 sub-
stituent were converted to
a-azido acids and then in four steps
Herein we describe efforts to improve the potency and pharma-
cokinetic (PK) properties of tetrafluorophenylmethyl ketone inhi-
bitors derived from 4. In vivo PK profiling of 4 in mice revealed
moderate half-life and clearance values and reasonable bioavail-
ability. These in vivo data were correlated with in vitro ADME sur-
rogates to guide an optimization strategy focused on reducing
lipophilicity while maintaining or improving upon the in vitro bio-
chemical and antiparasitic activity. Structure-aided design was
employed to select P1–P3 side chains that would retain key
hydrophobic and hydrogen bonding interactions while reducing
overall lipophilicity (calculated as AlogP in Vortex, Dotmatics).
Guided by this improved analogs such as 21 were developed that
exhibit improved anti-trypanosomal activity in vitro, combined
with superior oral exposure, half-life, and bioavailability as com-
pared to 4.
The in vivo pharmacokinetic properties of 4 were evaluated to
establish a baseline for further optimization work. In vitro ADME
parameters were also determined in the hope that in vitro surro-
gates could be correlated with key in vivo parameters. The
in vivo PK profile of 4 in mice turned out to be quite reasonable
as a starting point for further optimization. Hence, the compound
exhibits a reasonably long half-life in mice (T1/2 = 3.3 h), moderate
clearance (CL = 36.2 mL/min/kg), and oral bioavailability of ꢀ20%.
A steady-state volume of distribution (Vss) of 4.2 L/kg suggested
good tissue penetration, as is desirable for a Chagas’ therapeutic.15
to the tetrafluorophenylmethyl azido ketone intermediates A
(Scheme 1). Propargyl amines bearing the desired P2 substituent
were prepared in non-racemic form using Ellman’s chiral sulfi-
namide auxiliary.16 The amines were next subjected to reductive
amination with heterocyclic aldehydes or alternatively were cou-
pled to heteroaryl carboxylic acids to afford intermediates B bear-
ing the desired P2 and P3 substituents. Finally, copper(I)-catalyzed
azide–alkyne cycloaddition (CuAAC) reaction between intermedi-
ates A and B afforded the final analogs 4-23.
The new analogs were tested for cruzain inhibition using a bio-
chemical assay, as described previously.17 To facilitate the rapid
evaluation analogs, we determined IC50 values rather than full
kinetic parameters. While kinact/Ki values are generally preferred
when evaluating irreversible inhibitors, IC50 values can provide
useful rank-order SAR, provided that pre-incubation times are con-
sistent. The assay was performed with a final cruzain concentra-
tion of 0.1 nM in a pH 5.5 assay buffer comprising 100 mM
sodium acetate, 5 mM DTT, 0.01% Triton X-100 and 10 mM EDTA.
N
N
N
O
F
F
X
NH
P3
O
F
F
P2
P1
compounds 4-23
X = CH2 or C=O
Like 1, compound 4 inhibits CYP3A4 in vitro in the low
lM regime
(CYP 3A4 IC50 = 3.8 M). Despite being highly lipophilic
l
O
O
(AlogP = 7.0) compound 4 was found to exhibit reasonable stabil-
ity to cultured liver microsomes, consistent with the long half-life
observed in mice (T1/2 ꢀ3.3 h). Permeability in an MDCK cell mono-
layer assay was modest-to-low and solubility was qualitatively
estimated to be low as well, both factors likely contributing to
the modest bioavailability observed. Thus, an initial target of the
optimization campaign was to reduce lipophilicity, with the expec-
tation that improvements in solubility and permeability would
contribute to greater bioavailability and overall exposure on oral
dosing.
O
N
S
P2
P1
N
N
N
N
S
O
N
Quin
Py
BnThia
P3
BnFur
PzPm
To improve both potency and in vivo exposure, we sought to
identify new P1–P3 moieties that would retain the hydrogen bond
Figure 2. Summary of the structural chemotypes explored in this work.
Neitz, R. Jeffrey
