Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

Article abstract of DOI:10.1016/j.bmcl.2018.03.020

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no trea

Full text of DOI:10.1016/j.bmcl.2018.03.020

Accepted Manuscript
Optimization of Dipeptidic Inhibitors of Cathepsin L for Improved Toxoplasma
gondii Selectivity and CNS Permeability
Jeffery D. Zwicker, Nicolas A. Diaz, Alfredo J. Guerra, Paul D. Kirchhoff, Bo
Wen, Duxin Sun, Vern B. Carruthers, Scott D. Larsen
PII:
S0960-894X(18)30199-9
https://doi.org/10.1016/j.bmcl.2018.03.020
BMCL 25674
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
26 January 2018
7 March 2018
Please cite this article as: Zwicker, J.D., Diaz, N.A., Guerra, A.J., Kirchhoff, P.D., Wen, B., Sun, D., Carruthers,
V.B., Larsen, S.D., Optimization of Dipeptidic Inhibitors of Cathepsin L for Improved Toxoplasma gondii
Selectivity and CNS Permeability, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.03.020
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Optimization of Dipeptidic Inhibitors of Cathepsin L for Improved Toxoplasma gondii Selectivity and
CNS Permeability
Jeffery D. Zwickera,b, Nicolas A. Diaza,b, Alfredo J. Guerrac, Paul D. Kirchhoffb, Bo Wend, Duxin Sund, Vern B.
Carruthersc, and Scott D. Larsena,b
aVahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
b Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
cDepartment of Immunology and Microbiology, University of Michigan, Ann Arbor, Michigan 48109, United States
dPharmacokinetics Core, Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States
*Corresponding author. Tel: 734-615-0454
E-mail addresses: sdlarsen@med.umich.edu
KEYWORDS: Toxoplasma gondii, toxoplasmosis, cathepsin L, protease, infectious disease, antiprotazoal agents, blood-brain-barrier,
central nervous system
_________________________________________________________________________
Abstract
The neurotropic protozoan Toxoplasma gondii is the 2nd leading cause of death due to foodborne illness in the US, and has been
designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment
options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L
(TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the
first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a
known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma
vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS
penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 M to as low as 110 nM and we successfully
demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step
toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.
________________________________________________________________________
With an estimated two billion people chronically infected, Toxoplasma gondii is one of the most pervasive
infectious agents in the world.1, 2 This neurotropic protozoan is the 2nd leading cause of death due to foodborne
illness in the US, and has been designated as one of five neglected parasitic infections targeted by the Center for
Disease Control and Prevention for public health action.3-5 In its mammalian hosts, T. gondii exists in two
principal forms, the tachyzoite and bradyzoite. The initial disease state is caused by the rapidly replicating
tachyzoite form. This stage typically lasts for a several days to weeks during which the parasite disseminates to
peripheral sites including the central nervous system (CNS). T. gondii then transitions into a chronic bradyzoite
cyst form within the CNS and muscle tissue to establish life-long infection.6, 7 This chronic stage infection was
once considered to be benign, but recent studies have identified associations with variety of host behavioral
changes and mental illnesses including schizophrenia, bipolar disorder, and obsessive compulsive disorder.8-12
In the case of immunocompromised hosts (e.g. chemotherapy patients and HIV-positive individuals), the
parasite cysts can reactivate into the active tachyzoite form, leading to more serious complications such as,
encephalitis, blindness, and death.1, 13, 14 Currently, there is no efficacious T. gondii therapeutic capable of
treating the chronic-phase infection. Given the limitations and current inadequacies of antiparasitic therapies,
there is a serious need for the development of novel and alternative strategies for the treatment of chronic T.
gondii infection.
1

Figure 1. Literature Lead Cathepsin L Inhibitors.
Inhibition of vital cysteine proteases is an increasingly attractive approach for the treatment of various
disease states caused by protozoans such as Trypanosoma cruzi, Plasmodium falciparum, and others.15-17
Recently, we demonstrated that T. gondii cathepsin protease L (TgCPL) is critical to parasite survival during the
19
chronic phase of infection.18,
Inhibition of TgCPL in tissue culture by the irreversible inhibitor
morpholinurea-leucine-homophenylalaninevinyl phenyl sulfone 1 (LHVS) (Figure 1) kills T. gondii cysts,
further implicating TgCPL as a viable target for treatment of the chronic stage infection. Unfortunately, LHVS
is unable to cross the blood brain barrier precluding its use as a proof-of-concept probe in infected mice.20
Additionally, LHVS is a nonselective and irreversible inhibitor, increasing the possibility of off-target effects
and poor selectivity across cathepsin isoforms.
An enormous amount of drug discovery and development has been done in regard to both human and
parasitic cathepsins isoforms, and extensive mechanistic and structural information is available.21-28 Given the
close homology between the human and parasitic isoforms of cathepsin L, we considered the vast libraries of
inhibitors already developed against human isoforms to be a promising source of lead anti-parasitic
compounds.29 To identify a better potential lead compound than LHVS, we conducted a broad literature search
of structurally diverse CPL inhibitors emphasizing physicochemical properties predictive of CNS permeability,
selectivity over other human cathepsins, and potency (IC50 < 1 µM).
Table 1. Comparison of calculated properties of lead inhibitors vs CNS and non-CNS drugs
Compound
MW
SlogP
tPSA
HBA
HBD
RotB
288 ± 88
383 ± 210
527.69
2.8 ± 1.4
2.1 ± 2.9
3.6
46 ± 26
98 ± 84
104.8
2.6 ± 1.3
1.0 ± 0.9
4.0 ± 2.6
6.2 ± 5.5
14
CNS drug
4.8 ± 4.1
2.8 ± 3.3
non-CNS drug
5
4
2
2
1
2
381.48
2.4
99.8
10
Calculated using The Molecular Operating Environment (MOE), version 2008.10, Chemical Computing Group Inc., Montreal,
Quebec, Canada.30 Mean values and standard deviations derived from 198 approved CNS drugs and 1015 approved non-CNS drugs
(DrugBank).
We ultimately selected our initial lead compound 2 (Figure 1) based on its calculated properties: molecular
weight (MW), topological polar surface area (tPSA), numbers of hydrogen bond acceptors and donors (HBA,
HBD), and rotatable bonds (RotB), which are collectively closer to marketed CNS drugs vs non-CNS drugs
than are those of LHVS and most other literature leads we considered (Table 1)31. In addition, we considered
the ease with which we could expect to improve predicted BBB access by further lowering the tPSA.
2

Figure 2. LIGPLOT (right) diagram generated in MOE, shows key interactions between 2 and HsCPL. Hydrogen bonds: blue arrows,
lipophilic interactions: blue spheres. Derived from PDB: 3HHA
As the first ever SAR campaign targeting TgCPL, we elected to use a dipeptide nitrile scaffold as our lead,
based on the extensive amount of dipeptide-type cathepsin inhibitors previously developed for related human
isoforms.32-34 We believed the use of dipeptide probes would enable rapid elucidation of the pharmacophore
necessary for the selective inhibition of TgCPL due to their synthetic accessibility and ease of diversity
introduction. Another key rationale for selecting 2 as our lead was the aminoacetonitrile “warhead”. The
majority of small molecule protease inhibitors incorporate a functional group that can react with the active site
cysteine or serine in a reversibly covalent manner that imparts high affinity with relatively low off-target
reactivity. 32, 35, 36 The aminoacetonitrile group has been extensively studied and is present in a number of FDA-
approved drugs and advanced clinical candidates, including vildagliptin, saxagliptin, and odanacatib.37
Furthermore, its low molecular weight and relatively low tPSA were considered advantageous for achieving
CNS permeability.
3

Figure 3. Comparison of 2 bound to HsCPL (A, C) and a superposition of 2 modeled into the active site of TgCPL (B, D)
depicting key non-conserved residues. A) HsCPL with key residues colored. B) TgCPL with key residues colored C)
Lipophilicity mapping of HsCPL (green: lipophilic; magenta: polar); D) Lipophilicity mapping of TgCPL. (Derived from
PDB codes: 3HHA and 3F75)
Finally, we selected 2 as a lead because of the availability of an X-ray co-crystal structure of it bound to
human cathepsin L (HsCPL, (PDB: 3HHA).31 Opportunely, a crystal structure of TgCPL was also available
from our previous work, which we expected would greatly facilitate the design of TgCPL-selective inhibitors.31,
38-41
The crystal structure of 2 bound to HsCPL shows that the active site Cys25 is covalently added to the
nitrile, consistent with other reversibly-covalent nitrile based inhibitors of cysteine proteases, and that the ligand
makes hydrogen-bonding interactions with Asp162, Gly68, and Gln19 (Figure 2).32, 35 Importantly, the bound
inhibitor 2 can be overlaid on our crystal structure of TgCPL to identify proximal structural differences between
18
the cathepsins that can be exploited to achieve selective inhibition of the T. gondii enzyme (Figure 3). In
particular this overlay reveals a smaller S2 pocket for the Toxoplasma cathepsin, as well as four non-conserved
residues (colored in Figures 3A,B). Further comparison of the protein sequence alignment of TgCPL and the
human cathepsin isoforms A-X, revealed that these key residues are either unique to TgCPL, or generally non-
conserved across human isoforms. The S2 pocket across the human cathepsins tends to be more uniformly
lipophilic (colored green in Figures 3C,D) than TgCPL, and the inclusion of a basic residue in the P2 position,
which we predicted might interact strongly with non-conserved Asp-218 in TgCPL, is generally not tolerated.
With the exception of human cathepsin B, which bears a Glu245 in this respective position, the overall topology
in the S2 subsite is thus substantially different than that of TgCPL.42 This provided excellent supporting
evidence that optimization in the P2 position would be paramount in an effort to gain selectivity over the bulk
of the human isoforms.
4

Our primary objectives in this preliminary work were: 1) to develop analogs with better predicted CNS
permeability without losing potency; and 2) to determine if we can begin to shift the selectivity of 2 away from
HsCPL and towards TgCPL. Herein we report the development of a series of dipeptide nitrile probes that help
to define pharmacophoric features key to improving both potency and selectivity for TgCPL, including one
analog with CNS permeability in vivo.
Scheme 1. General synthesis of dipeptide nitriles. Reagents and conditions: a) H2SO4, MeOH, reflux; b) HATU, DIPEA, DMF; c)
1N LiOH(aq), THF, MeOH; d) 2-aminoacetonitrile bisulfate, HATU, DIPEA, DCM, 0°C-rt
Synthesis of analogs 7a-r (Scheme 1) was performed analogous to the literature precedent for the peptide-
nitrile cathepsin inhibitors.31, 32, 34, 41 We began with the esterification of various commercially available L-
amino acids 3a-h to afford their respective methyl esters 4a-h. Subsequent amide coupling to the appropriate
aryl acids gave 5a-r. The methyl ester was then saponified under mild conditions with LiOH in a THF:MeOH
system to afford the desired free acid 6a-r. Initial coupling with 2-aminoacetonitrile gave several unwanted side
reactions and generally low yields when using DMF or THF as the solvent, regardless of the temperature or
coupling reagent used. Upon changing to dichloromethane we found that, while solubility was not always ideal,
the desired products 7a-r were formed cleanly and in moderate yields.
5

Scheme 2. Synthesis of dimethyl-lysine analog. Reagents and conditions: a) 4-Fluorobenzoic acid, HATU, DIPEA, DMF, 98%; b)
4N HCl/dioxane, 99%; c) formaldehyde, sodium triacetoxyborohydride, MeOH, 32%; d) 1N LiOH(aq), THF, MeOH, 100%; e) 2-
aminoacetonitrile bisulfate, HATU, DIPEA, DCM, 0°C-rt, 11%.
Synthesis of 13 was achieved as shown in Scheme 2. Nε-Boc-L-lysine methyl ester hydrochloride 8 was
coupled to 4-fluorobenzoic acid to provide intermediate 9. Boc removal from 9 afforded the HCl salt of 10
which was immediately followed by dimethylation of the sidechain to provide compound 11. Saponification of
11 with lithium hydroxide provided 12, and subsequent amide coupling using HATU gave the desired product
13.
Scheme 3. Reagents and conditions: a) Leucine hydrochloride, K2CO3, MeOH, 50°C, 18h; b) Zn(BH4)2, ACN/DME, -40°C, 5h, 87%;
c) NaBH4, THF, 93%; d) 2-aminoacetonitrile bisulfate, or 1-aminocyclopropane-1-carbonitrile HCl, HATU, DIPEA, DCM, 0°C-rt,
45-89%
Trifluoroethyl amine analogs (18a-b, 19) were generally synthesized as shown in Scheme 3 following
previously established methods in the development of odanacatib.43, 44 Synthesis began with the condensation of
the trifluoroacetophenone 14 and L-Leucine-methylester under basic conditions to afford the imine intermediate
15. The R,S diastereomer 17, was obtained via dropwise addition of NaBH4 dissolved in THF at -40°C in a 10:1
dr. Alternatively the (S,S) diastereomer could be obtained through a chelation controlled reduction of the imine
with zinc borohydride in acetonitrile/dimethoxyethane at -40°C, providing 16 in an 11:1 dr. The (S,S) and (R,S)
compounds 18a, 18b, and 19 were obtained in 45-94% yield through the previously described amide coupling
conditions (Scheme 1).
Scheme 4. Reagents and conditions: a) 4-fluorobenzoic acid, HATU, DIPEA, DMF, 34%; b) Selectfluor, Fe2(SO4)3, NaBH4,
acetonitrile:water, 0°C-rt, 27%; c) 1N LiOH(aq), THF:EtOH, 0°C-rt, 100%; d) 2-aminoacetonitrile bisulfate, HATU, DIPEA, DCM,
0°C-rt, 18%
6

Fluoroleucine analog 24 was prepared as shown in Scheme 4 through a sequence derived from previously
reported methods for analogous compounds.44-47 Dehydroleucine 20, as the CSA salt, was coupled to 4-
fluorobenzoic acid to provide intermediate amide 21. Initially, we tried to achieve fluorination of 21 using a
variety of the previously reported conditions, predominantly using reagents that are a source of nucleophilic
fluorine such as HF:Urea or HF:Pyridine. However, these conditions generally suffered from poor yields and
unwanted side reactions such as cyclization or elimination. We then opted to try an electrophilic source of
fluorine as an alternative approach, derived from that reported by Fanelli et. al..48 Activation of the olefin with
Fe(III), followed by the sequential addition of Selectfluor and NaBH4 provided the desired product 22 in
moderate which, was then saponified to 23 and directly coupled to 2-aminoacetonitrile, affording the desired
compound 24.
All new compounds were evaluated in vitro for inhibition of both TgCPL and HsCPL activity by monitoring
the rates of hydrolysis of Z-Leu-Arg-7-amino-4-methylcoumarin (Z-L-R-AMC). Results are summarized in
Tables 2-5. We first re-synthesized literature lead 2 to benchmark its relative potency against TgCPL versus
HsCPL. In vitro testing revealed low micromolar potency against TgCPL, and 46-fold more potent inhibition
against HsCPL (Table 2).
Initially we replaced the pyrazole heterocycle in the P3 position of 2 with phenyl in order to remove two
nitrogen atoms (7b) and rapidly lower the tPSA of 2, thereby increasing the potential for CNS permeability.
This exchange resulted in a 2-3-fold drop in potency against HsCPL while enhancing activity against TgCPL,
improving the TgCPL-selectivity about 4-fold. The t-butyl group of 7b was then replaced by trifluoromethyl to
stabilize the now electron-rich P3 aromatic ring to potential oxidation by CYP450 enzymes, giving 7a. This
analog lost potency against TgCPL, but maintained effectiveness versus HsCPL. Since both of these analogs
had reduced selectivity for HsCPL compared to lead 2 we retained both of the P3 groups during our initial
survey of P2.
Table 2. Cathepsin L inhibitory activity of initial P2 and P3 analogs.
TgCPLa IC50 HsCPLb IC50
Selectivity
for HsCPL c
0.7
Name
1
Ar
R
(µM)
(µM)
N/A
N/A
0.002
0.003
2.0
2.4
0.044
0.11
46.4
22.5
2
7a
7

1.4
1.4
0.13
11.3
33.5
7b
7c
0.042
1.2
6.1
4.6
0.092
0.086
0.24
13.3
71
7d
7e
7f
18.8
0.12
0.12
0.077
0.32
1.9
0.4
7g
7h
a,bIC50 for Toxoplasma gondii Cathepsin L (TgCPL) or human Cathepsin L (HsCPL). Values are mean of at least 3 independent
c
experiments. Selectivity ratio for human Cathepsin L as defined by TgCPL IC50/HsCPL IC50.
The vast majority of selectivity between human cathepsin isoforms comes from the P2-S2 interaction.49 The
S3 and S1 subsites also play significant roles in inhibitor selectivity, while the S’ subsites (toward the C-
terminus of the scissile bond) are highly conserved between isoforms and generally difficult to exploit.22
Inspection of the cathepsin crystal structures revealed several active site residues unique to the T. gondii
isoform vs the human isoform that could potentially be exploited to gain selectivity and potency. As noted
above, the TgCPL S2 site contains a unique aspartate residue (Asp218), as opposed to the human isoform,
which bears a hydrophobic alanine in this position (colored green in Figures 3A, B). Overlaying TgCPL with
the co-crystal structure of our lead compound bound to HsCPL indicates that the 3-methylphenylalanine (3-Me-
Phe) core of 2 sits approximately 3-4 Å from this key aspartate (Figure 3B). Due to the proximity of the core to
Asp218 we hypothesized we might be able to build in a potential H-bond to improve potency and selectivity.
8

The P2 3-methylphenylalanine residue of analogs 7a and 7b was thus replaced with 3-hydroxyphenyalanine
or 3-methoxyphenylalanine (7c-7f) in an attempt to pick up a selective hydrogen bonding interaction with the
non-conserved Asp218 of TgCPL. We had hoped this would provide some initial gain in selectivity toward the
parasite cathepsin, but the results were inconsequential changes in potency. This might be due to the rigidity of
the Asp218 backbone orienting the residue away from the S2 pocket and preventing a beneficial interaction.
Figure 4. Overlay of 2 (gray) and 18b (orange) into the crystal structure of TgCPL (PDB code: 3F75) depicting clashes with 2 in the
S2 pocket. Hydrogen bonds and clashes are represented as blue and orange dashed lines, respectively. Pocket surface coloration
represents polar (purple), lipophilic (green), and solvent exposed (red) areas.
Molecular modeling also identified several predicted clashes with HsCPL when the larger phenylalanine
side chains are in the P2 position (2 in Figure 4). We were pleased to discover that replacement of the entire
aromatic P2 residue with a less bulky leucine (7g,7h) significantly reduced selectivity for HsCPL, consistent
with the smaller S2 subsite in the TgCPL as compared to the human isoform (Table 2). With this single change,
we reduced the selectivity of 2 for HsCPL from 10-20 fold to <2 fold. This is an excellent validation that the
differences in the S2 pockets can be exploited for improving TgCPL selectivity.
Table 3. Inhibitory activity of additional aryl P3 substitution analogs.
TgCPLa IC50 HsCPLb IC50
Selectivity
Name
7i
Ar
(µM)
(µM)
for HsCPL c
0.35
0.11
3.3
0.18
0.015
11.7
7j
9

0.36
0.54
0.048
0.28
7.6
1.9
7k
7l
0.53
0.25
0.14
0.28
3.8
0.9
7m
7n
a,b,cSee Table 2 for heading definitions.
We then returned to optimization of the P3 position in an effort to reduce the molecular weight of 7h while
retaining a halogenated pendant for metabolic stability (Table 3). Removal of the trifluoromethyl from the P3
pendant to afford the simple phenyl (7i) unfortunately reversed the selectivity back to HsCPL over TgCPL. A
similar trend was observed with analogs 7j, 7l, and 7m, which bear a 3-methyl, 2-chloro-4-fluoro, or 2,4-
dichloro substitution pattern, respectively, on the P3 pendant. Replacement of the 3-trifluoromethyl of 7h with
a fluorine (7k) also resulted in a large drop in potency for TgCPL and an increase for the human isoform. The 4-
fluoro analog 7n, however, was equipotent at TgCPL and HsCPL, and lowered the MW below 300, making this
a more promising candidate for CNS permeability as compared to the somewhat more potent and TgCPL-
selective 4-trifluoromethyl analog 7h.
Table 4. Inhibitory activity of additional aliphatic P2 analogs
TgCPLa IC50 HsCPLb IC50
Selectivity
Name
7o
R
(µM)
(µM)
for HsCPL c
0.42
0.19
2.3
5.7
6.6
14
1.2
7p
13
816
0.02
10

4.0
2.0
1.2
9.8
2.5
1.2
3.5
4.1
1.6
1.6
0.3
2.4
24
25
7q
7r
a,b,cSee Table 2 for heading definitions.
We then explored a dimethyl lysine at P2 as a basic group to establish a salt bridge with the non-conserved
Asp218 of TgCPL (13 in Table 4). While this was predicted by modeling to be the optimal length to interact
with the S2 aspartic acid, it nevertheless resulted in a major loss in potency against both isoforms. However, it
should be noted that the loss in potency was significantly greater against the human isoform, demonstrating that
despite the S2 pocket of these two enzymes being very similar, subtle differences at the P2 residue can offer
drastic changes to the selectivity profile. Further exploration of basic residues at P2 is therefore warranted in
future SAR studies.
Figure 5. SMARTCyp prediction of the top three sites of metabolism.
Cyclohexyl P2 analogs have been reported as inhibitors of human protease inhibitors that offered some
improvement in either binding or metabolic stability.50 To further probe aliphatic SAR at the TgCPL P2
position, both were made (7o and 7p,Table 4); however, neither offered any improvement. Rather, a decrease in
potency and TgCPL selectivity was observed for both, more drastically in the case of 7p, which is a closer
mimic of valine than leucine. Substitutions much larger than leucine were thus apparently not well tolerated in
TgCPL, consistent with the poor activity we had observed earlier with our phenylalanine analogs, so we
attempted to install isosteres that were closer in size to leucine. While the use of fluoroleucine to improve
metabolic issues has been successfully demonstrated in the development of odanacatib51, we were surprised to
see a drastic drop in potency against both TgCPL (IC50= 4.05 µM) and HsCPL (IC50= 2.51 µM) with compound
24. Similarly, exchange of the P2 leucine with either a neopentylglycine, or dehydroleucine (7q, 25) resulted in
a significant loss of potency (IC50= 1.19 µM and 2.00 µM respectively). Surprisingly, even the cyclopropyl
analog 7r showed a large drop in potency as compared to 7n. While these structural changes are minor, the
substantial changes in potency clearly demonstrate the high level of sensitivity of the P2 position to steric
modification. Within this initial set of dipeptide nitrile TgCPL inhibitors, leucine thus proved to be the most
optimal residue in the P2 position for both potency and reduced selectivity for HsCPL.
11

Table 5. Inhibitory activity of trifluoroethylamine and cyclopropyl-nitrile analogs.
TgCPLa IC50 HsCPLb IC50
Selectivity
Name
7n
R
(µM)
(µM)
for HsCPL c
0.25
0.28
0.9
0.66
0.11
0.40
0.16
0.59
0.093
0.14
1.1
1.2
2.9
1.9
19
18a
26
0.084
18b
See Table 2 for heading definitions.
Most CNS drugs do not tolerate more than 1 or 2 hydrogen bond donors. As shown in the LIGPLOT
diagram from the co-crystal structure of HsCPL and compound 2 (Figure 2), the carbonyl of the P2-P3 amide is
not actively engaged by the enzyme and therefore may be unnecessary. Replacement of this amide with the
well-established trifluoroethlyamine bioisostere offered an effective way to reduce our overall tPSA, greatly
enhancing our chances for CNS penetration.43, 51-53 We found that the (R,S) diastereomer 19 gave a substantial
drop in overall potency for both cathepsins (Table 5). The (S,S) diastereomer 18a, however, doubled the
potency against both the Toxoplasma and human enzymes, providing the most potent nitrile-based inhibitor to
date against TgCPL with an IC50= 110 nM. This diastereomeric preference is consistent with literature reports
of other dipeptide cathepsin inhibitors incorporating the trifluoroethyl amine bioisostere.43
Table 6. Metabolic stability of select analogs in mouse liver microsomes.
MLM
t1/2 (min)
Compound
R1
3-CF3
4-F
R2
37
7h
7n
>60
12

4-F
4-F
4.4
22
18a
18b
The metabolic stability of our dipeptide nitrile series was initially evaluated in mouse liver microsomes
(MLM) to assess suitability for future in vivo studies and to further guide our design. Compound 7h was
evaluated to benchmark the dipeptide nitrile series (Table 6). We observed that compound 7h had a reasonably
long half-life (37 min). This parameter was further improved to >60 min with less lipophilic compound 7n,
despite several metabolic liabilities predicted by SMARTCyp (Figure 5).54 Interestingly, while replacement of
the P2 amide with a trifluoroethylamine (18a) removed an amide that could be a metabolic liability, the half-life
in mouse liver microsome decreased to 4.4 minutes. It is likely that the increased lipophilicity simply enhanced
binding to metabolizing enzymes. Two of the most likely positions for metabolism of 18a are oxidation alpha to
the nitrile warhead and hydrolysis of the P1 amide bond.54 The incorporation of a cyclopropyl at the P1 position
serves to block this site from oxidation and to slow hydrolysis of the P1 amide, a strategy successfully used in
the development of odanacatib and other inhibitors of human cathepsins.51, 53, 55 Cyclopropyl analog 18b indeed
provided a significantly improved MLM half-life of 22 min, affording a compound more suitable for further in
vivo analysis. It also retained potency for TgCPL, which is consistent with the predicted projection of the
cyclopropyl group at P1 out into solvent (Figure 4).
Table 7. In Vivo Exposure of Compound 18b Following IP Administration to Micea
30 min
110
2 h
138
4 h
10
7 h
Time Post-Dose
Plasma Conc. (ng/mL)
BLQ
BLQ
Brain Conc. (ng/g)
54
BLQ
BLQ
a
CD-1 mice were injected intraperitoneally with a single 10 mg/kg dose. The data shown are mean values from 2 mice at each time
point. BLQ = Below Limit of Quantification.
Compound 18b was advanced to an abbreviated in vivo pharmacokinetic study in mice (Table 7). Our main
objective was to determine if this chemotype could penetrate the CNS. Significantly, we were able to detect
compound in the brain, with a brain/plasma ratio of 0.5 at 30 minutes. Unfortunately, the overall levels in both
brain and plasma were quite low, despite the fact that 18b was shown to have good aqueous solubility (630 µM)
and excellent plasma stability (~100% parent remaining after 24h). The plasma levels and rapid disappearance
indicates that this compound has a high volume of distribution, high clearance or both. Studies are currently
underway to address this issue and to develop new analogs with improved pharmacokinetic profiles.
In summary, we have demonstrated for the first time that it is possible to modify a well established
chemotype for inhibition of human cathepsins (dipeptide nitrile) and shift selectivity away from the human
isoform and towards the Toxoplasma gondii isoform, a novel target for toxoplasmosis. Furthermore, we have
demonstrated that the physical properties of the dipeptide can be adjusted to be more favorable for CNS
penetration without loss of activity, particularly through reduction in tPSA and number of HBA. Key analogs in
this campaign are summarized in Figure 6. Initially, our lead compound 2 was approximately 46-fold selective
for HsCPL over TgCPL. We developed several compounds that either displayed equipotency for HsCPL and
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TgCPL (7n), or exhibited slight selectivity (up to 3-fold) for TgCPL over HsCPL (7h,7q). Notably, compound
13 (Table 4) demonstrates the potential for achieving outright selectivity for TgCPL through the incorporation
of a basic residue in the P2 position, an avenue we are exploring in future studies. Furthermore, we improved
the potency of our inhibitors for the T. gondii enzyme from 2 µM to as low as 115 nM. Although we
successfully demonstrated that compound 18b can penetrate the blood brain barrier, it also exhibits rapid
clearance in vivo. Current endeavors in our lab are aimed at further defining the pharmacophore for TgCPL via
optimization of the P3 and P2 positions. Additionally, we are improving the pharmacokinetic profile for these
inhibitors, so they may be advanced to in vivo murine infection studies in order to establish proof-of-concept
that TgCPL is a viable target for treating the chronic form of toxoplasmosis.
Figure 6. Key compounds in the optimization of 2 for TgCPL potency, metabolic stability, and a CNS-penetrant profile.
Acknowledgements
This work was supported by NIAID of the National Institutes of Health under award number
R21/R33AI127492 (V.B.C. and S.D.L.). Its contents are solely the responsibility of the authors and do not
necessarily represent the official views of the NIH. V.B.C. and S.D.L. were also supported by a grant from the
Center for Discovery of New Medicines, University of Michigan. J.D.Z. acknowledges support from the
University of Michigan Pharmacological Sciences Training Program (PSTP) training grant (T32-GM007767),
and the Rackham Merit Fellowship (RMF). N.A.D. received support from the National Science Foundation
(NSF DBI – #1263079), and National Institutes of Health (NIH/NIGMS GM007717).
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Graphical Abstract:
Optimization of Dipeptidic Inhibitors of
Cathepsin L for Improved Toxoplasma gondii
Selectivity and CNS Permeability
Leave this area blank for abstract info.
Jeffery D. Zwicker, Nicolas A. Diaz, Alfredo J. Guerra, Paul D. Kirchhoff, Bo Wen, Duxin Sun, Vern B.
Carruthers, and Scott D. Larsen
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