Discovery and Optimization of Triazine Nitrile Inhibitors of Toxoplasma gondii Cathepsin L for the Potential Treatment of Chronic Toxoplasmosis in the CNS

Article abstract of DOI:10.1021/acschemneuro.9b00674

With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains one of the most pervasive and infectious parasites. Toxoplasma infection is the second leading cause of death due to foodborne illness in the United States, causes severe disease in immunocompromised patients, and is correlated with several cognitive and neurological disorders. Currently, no therapies exist that are capable of eliminating the persistent infection in the central nervous system (CNS). In this study we report the identification of triazine nitrile inhibitors of Toxoplasma cathepsin L (TgCPL) from a high throughput screen and their subsequent optimization. Through rational design, we improved inhibitor potency to as low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold for TgCPL versus human isoform), and improved metabolic stability (t1/2 > 60 min in mouse liver microsomes) guided by a metabolite ID study. We demonstrated that this class of compounds is capable of crossing the blood-brain barrier in mice (1:1 brain/plasma at 2 h). Importantly, we also show for the first time that treatment of T. gondii bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite viability with efficacy equivalent to a TgCPL genetic knockout.

Full text of DOI:10.1021/acschemneuro.9b00674

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Article
Discovery and Optimization of Triazine Nitrile Inhibitors of Toxoplasma gondii
Cathepsin L for the Potential Treatment of Chronic Toxoplasmosis in the CNS
Jeffery D. Zwicker, David Smith, Alfredo J. Guerra, Jacob R Hitchens, Nicole Haug, Steve Vander Roest, Pil
Lee, Bo Wen, Duxin Sun, Lu Wang, Richard Keep, Jianming Xiang, Vern B. Carruthers, and Scott D Larsen
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00674 • Publication Date (Web): 06 Feb 2020
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Discovery and Optimization of Triazine Nitrile Inhibitors of
Toxoplasma gondii Cathepsin L for the Potential Treatment of
Chronic Toxoplasmosis in the CNS
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Jeffery D. Zwicker†,ǂ, David Smith‡, Alfredo J. Guerra‡, Jacob R. Hitchens†,ǂ, Nicole Haug‡, Steve Vander
Roest#, Pil Lee†,ǂ, Bo Wen§, Duxin Sun§, Lu Wang§, Richard F. Keep+, Jianming Xiang+, Vern B.
Carruthers‡, and Scott D. Larsen*†,ǂ
†Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor,
Michigan 48109, United States
ǂDepartment of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
‡Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109,
United States
#Center for Chemical Genomics, Life Sciences Institute, Ann Arbor, MI 48109, United States
§Pharmacokinetics Core, Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor,
Michigan 48109, United States
+Department of Neurosurgery, University of Michigan, Ann Arbor, Michigan 48109, United States
*Corresponding author. E-mail address: sdlarsen@med.umich.edu
KEYWORDS: Toxoplasma gondii, antiparasitics, toxoplasmosis, cathepsin L, triazine nitrile, protease,
infectious disease, antiprotazoal agents, blood-brain barrier, central nervous system
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Abstract: With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains
one of the most pervasive and infectious parasites. Toxoplasma infection is the 2nd leading cause of death
due to foodborne illness in the US, causes severe disease in immunocompromised patients, and is
correlated with several cognitive and neurological disorders. Currently, no therapies exist capable of
eliminating the persistent infection in the central nervous system (CNS). In this study we report the
identification of triazine nitrile inhibitors of Toxoplasma cathepsin L (TgCPL) from a high throughput
screen, and their subsequent optimization. Through rational design, we improved inhibitor potency to as
low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold
for TgCPL versus human isoform), and improved metabolic stability (t1/2 > 60 min in mouse liver
microsomes) guided by a metabolite ID study. We demonstrated this class of compounds is capable of
crossing the blood-brain barrier in mice (1:1 Brain:Plasma at 2 hr). Importantly, we also show for the first
time that treatment of T. gondii bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite
viability with efficacy equivalent to a TgCPL genetic knockout.
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______________________________________________________________________________
Graphical Abstract:
O
N
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S2
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scaffold-hop
following HTS
CF3
H
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CN
N
CN
N
H
F
S3
S3
O
F
S2
2
4
TgCPL IC50 : 160 nM
HsCPL IC50 : 84 nM
HBD: 2, RotB: 8
TgCPL IC50 = 38 nM
HsCPL IC50 = 37 nM
HBD: 0, RotB: 7
Bradyzoite active: No
Bradyzoite acitve: Yes
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INTRODUCTION
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Estimates suggest over 30% of the global human population is currently infected with
Toxoplasma gondii, with approximately 60 million infected in the United States alone.1-3 Capable of
infecting nearly all warm-blooded mammals, this single-celled apicomplexan is the second leading cause
of death due to foodborne illness in the US.4 As a member of the phylum Apicomplexa, Toxoplasma
gondii exhibits a complex infection cycle in humans.5 Through consumption of contaminated food, or
direct exposure to cat fecal matter, the parasite enters an initial tachyzoite infection stage, in which the
parasite undergoes rapid asexual replication and disseminates throughout the host tissues, including the
central nervous system (CNS).6 Upon activation of the host immune response, T. gondii transforms into
its chronic bradyzoite phase, in which the parasite exists as lifelong tissue cysts within the muscle and
CNS. If the host maintains a healthy immune system, this chronic infection generally presents few
symptoms, although recently there is a growing body of evidence suggesting that this chronic stage
infection may significantly contribute to a wide array of neuropsychiatric symptoms including
schizophrenia, bipolar disorder, obsessive compulsive disorder, and various other mood alterations. 7-10
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A more obvious impact on human health from this parasite is revealed when the host immune
system is compromised. The unchecked parasite cysts reactivate into the acute tachyzoite form, causing
tissue inflammation and more serious complications such as: encephalitis, blindness, and death.11
Treatment options for toxoplasmosis reactivation are generally pyrimethamine in combination with
sulfadiazine, or clindamycin.12 However, these options are prone to adverse side effects and only act upon
the parasite in the peripheral system. Currently, no approved treatment options exist to clear the latent and
chronic Toxoplasma infection in the CNS, therefore it is critical to develop new and alternative treatment
strategies. A recent review by Deng et al13 summarizes current approaches to targeting enzymes critical to
the life cycle of the parasite, including calcium-dependent protein kinase 1 (TgCDPK1), thymidylate
synthase-dihydrofolate reductase, enoyl-acyl carrier protein reductase, and cathepsin L. TgCDPK1
inhibitors in particular have shown promise for treating the chronic form of T. gondii.14
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Cathepsins L and B are the major cysteine proteases found in protozoans, and are emerging as
viable targets for anti-parasitic agents due to their essential roles in parasite survival.15-17 For example,
Rhodesain (T. brucei), cruzain (T. cruzi), and falcipain (P. falciparum) express cathepsin L-like proteases
that have gained a considerable amount of attention for the potential treatment of African sleeping
sickness, Chagas disease, and malaria respectively.18 Toxoplasma gondii expresses five members of the
C1 family of cysteine proteases, including TgCPL, TgCPB, and TgCPC1-3.19-22 TgCPL is localized in the
plant like-vacuole (PLV) vacuolar compartment (VAC, used hereafter), and is also responsible for the
maturation and activation of TgCPB.20, 23 Although inhibition or disruption of TgCPL in the parasite has
been shown to moderately impede parasite invasion and growth in the tachyzoite stage of infection,
TgCPL is not critical to the acute stage.21, 23 Interestingly, parasites in the cystic bradyzoite stage express
heightened levels of TgCPL and TgCPB, suggesting proteolysis in the VAC plays an important role in the
chronic infection.24 Accordingly, bradyzoites deficient in TgCPL die after forming cysts in both culture
and in the neuronal cells of infected mice. Upon restoration of catalytically active TgCPL, bradyzoite
viability is re-established. In contrast, expression of a catalytically inactive TgCPL fails to restore cyst
viability. Taken together, these strongly implicate TgCPL as a viable target for the treatment of this
chronic stage infection. The parasite dependence on TgCPL has been further validated in vitro, as well as
through the treatment of cysts with the covalent, irreversible cathepsin inhibitor LHVS (1, Figure 1).25
This disruption of the proteolytic activity in the lysosomal VAC ultimately results in parasite death.26
However, LHVS is not a viable pharmaceutical lead as it failed to reduce neural cysts in infected mice,
due to its poor drug-like properties (e.g., fails to cross the blood-brain barrier).27
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Figure 1. Inhibitors of Toxoplasma gondii cathepsin L
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Recently, we reported the first lead optimization study for the inhibition of TgCPL with a series
of dipeptide nitriles.28 While we were able to gain insight into the pharmacophore necessary for TgCPL
inhibition and significantly improve the potency and selectivity over human isoforms, we were unable to
carry forward the dipeptide series due to the inadequate pharmacokinetic (PK) properties of optimized
lead 2. Despite being potent in vitro and demonstrably CNS penetrant, the drug was rapidly cleared in
vivo. While the dipeptide-nitriles are synthetically convenient for investigating SAR in the cathepsin
pockets, they have less than desirable physiochemical properties for gaining robust blood-brain barrier
(BBB) permeability, such as several rotational bonds, extensive hydrogen bonding, and high topological
polar surface area (TPSA).29 We thus began searching for alternative scaffolds that might exhibit better
physiochemical properties to improve upon the in vivo PK, while maintaining the pharmacophoric
elements necessary for potent TgCPL inhibition.
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Figure 2: Structural overlay and active sites of apo TgCPL (Cyan; PDB-3F75) and apo HsCPL (Orange
PDB-5MAJ)
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A high throughput screen for TgCPL inhibitors was conducted at the Center for Chemical
Genomics at the University of Michigan on over 150,000 small molecules. From this we identified
several inhibitor classes that demonstrated promising inhibitory activity against TgCPL, reasonable
synthetic approachability, and physiochemical profiles optimal for use as a lead scaffold in a CNS
targeted campaign. To minimize potential PK and off-target toxicity issues in later development, we
chose to exclude compounds that were peptidic in structure, or irreversibly covalent. After our triage
process had identified several potential lead molecules, we searched the literature to determine if any of
these chemotypes had any previously demonstrated activity against cysteine proteases. An extensive
amount of study has been done on inhibitors of human cathepsin L (HsCPL).30 Since Toxoplasma gondii
cathepsin L shares high homology with HsCPL, we considered chemotypes with precedent for inhibiting
HsCPL as good potential leads. The overall sequence similarity between the two is about 50 %. However,
for residues within 4.5 A of the triazine, the similarity is about 72 %. Only 5 out of 18 residues in the
binding site are different in the two structures (Figure 2). Among the several potential lead molecules
discovered in our screen, we selected a modestly active triazine nitrile 3 (TgCPL IC50 = 2.5 µM, HsCPL
IC50 = 2.3 µM) as a lead candidate (Figure 1), given its fragment-like size and solid literature precedent
as a cathepsin inhibitor class. Several recent publications have reported triazine nitrile-based inhibitors of
human cathepsins, and additionally demonstrated potent inhibition of rhodesain and falcipain.31-33 The
activity reported against these analogous cysteine proteases from the related apicomplexan parasites P.
falciparum and T. brucei further supports the potential of this chemotype as an antiparasitic agent.
Additional triazine nitriles, pyrazolopyrimidine nitriles, and related heterocylic-nitrile motifs have also
been reported as inhibitors of cysteine proteases.34-37
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Although these recent studies have established the potential of this chemotype as a clinically relevant
antiparasitic agent, Toxoplasma gondii, as well as other protozoan parasites such as T. brucei, establish a
chronic infection in the CNS. Compounds intended for CNS targets have a somewhat constrained
physiochemical profile as compared to peripheral drugs. Key among these are the minimization of both
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the number of hydrogen bond donors and rotatable bonds, which can increase the rate of passive BBB
penetration. Additionally, CNS drugs exhibit relatively low MW (<450), high lipophilicity (logP 2-5), and
exclusion of acidic moieties. The triazinyl-nitrile scaffold was particularly promising in that it could
readily incorporate the structural motifs necessary for binding in the S2 and S3 pockets of TgCPL (Figure
2), which we elucidated in our previous dipeptide work. This scaffold also has a potentially improved
pharmacokinetic profile and CNS permeability by reducing the rotational degrees of freedom, eliminating
a hydrolyzable amide and removing two hydrogen bond donors (HBD). Molecular docking and energy
minimization of the dipeptide nitrile (2) and proposed triazine nitrile (4) in the active site of TgCPL
showed good structural agreement into the pharmacophore of TgCPL inhibition (Figure 3). Altogether
this encouraged us to attempt a scaffold-hop from the optimized dipeptide to a triazine nitrile. In this
paper we report our success at this transformation, and our initial SAR work at enhancing selectivity for
the parasite enzyme vs human cathepsin L. Furthermore, we demonstrate for the first time the ability of
this class of compounds to inhibit bradyzoite viability in vitro and to penetrate into the brain in vivo, two
key milestones for developing an effective therapeutic for the chronic stage of T. gondii.
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HBD= 2
RotB= 8
HBD= 0
RotB= 7
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Figure 3. Predicted scaffold hop from optimized dipeptide nitrile to a
triazine nitrile. A) Predicted projection of common vectors into the S2 and
S3 sites of TgCPL based on computational docking of the dipeptide scaffold 2
and a corresponding triazine nitrile scaffold 4 into the crystal structure of
TgCPL derived from PDB: 3F75. B) Molecular overlay of dipeptide nitrile (2,
yellow) and triazine nitrile (4, teal) demonstrating good structural alignment.;
HBD= Hydrogen bond donor; Rot B= rotatable bonds;
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RESULTS AND DISCUSSION
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Synthesis of TgCPL inhibitors
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Scheme 1. General synthesis of triazine nitrile inhibitors
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Cl
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Int-1a
Int-2a
Int-3a
Int-4a
4, 49, and 42
Reagents and conditions: a) R2NH2, NaBH4, DCM, rt, or Titanium (IV) isopropoxide, Na(OAc)3BH,
DCM, 0 °C-rt, O/N; b) cyanuric chloride, DIPEA, DCM, -10 °C, 1-2 hr; c) morpholine, DIPEA, DCM,
0 °C-rt, 4 hr; d) KCN, DMSO:H2O (9:1), 80 °C, O/N.
Under anhydrous conditions, the desired aryl aldehyde or acetophenone Int-1a was condensed
with the desired alkylamine overnight, at which point the formed imine was reduced with sodium
borohydride or sodium triacetoxyborohydride (Scheme 1). Next, the reaction between the secondary
amine Int-2a and cyanuric chloride provided intermediates Int-3a. Subsequent SNAr with morpholine
afforded intermediates Int-4a. In the case of 50, oxetanamine was used as the P1 solubilizing group in
place of morpholine. Finally, cyanation of the scaffold with potassium cyanide in DMSO provided the
desired final compounds.
Scheme 2. Alternative general synthesis of triazine nitrile inhibitors
Route B
O
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Int-3b
Int-4b
65 examples
Reagents and conditions: a) i. Cyanuric chloride, DIPEA, DCM, -10 °C, 1-2 hr; ii. Morpholine,
DIPEA, DCM, 0 °C-rt, 4 hr; b) KCN, DMSO:H2O (9:1), 80 °C, O/N; c) NaH, DMF, 0 °C 30 min, then
ArCH2X when X=Br, Cl, Ts, or Ms; d) TFA:DCM (1:2), rt, 0.5-1 hr.
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To expedite rapid production of diverse analogs, a synthetic strategy analogous to that reported
by Giroud et. al. for the synthesis of triazine-nitrile HsCPL inhibitors was employed (Scheme 2).31 First,
cyanuric chloride underwent SNAr reactions with the desired benzyl or alkyl amines Int-1b to set the
various S2 and S3 vectors for intermediates Int-2b. A second SNAr step was employed to install a
morpholine as a P1 solubilizing group. Cyanation of the intermediate was performed with potassium
cyanide in DMSO, to afford Int-3b. Subsequently, the anion of intermediates Int-3b was generated using
sodium hydride, and the desired alkyl/benzyl halide was added to provide analogs Int-4b. In the case of
analogs bearing a Boc-protected amine side chain, TFA:DCM deprotection was employed to provide the
desired final compounds.
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Docking of the triazine scaffold into TgCPL and HsCPL: The IC50 for the triazine nitrile we
identified in our HTS (3) was benchmarked at 3.5 µM and 2.3 µM against TgCPL and HsCPL,
respectively (Table 1). Considering this lead bears an ethyl as the only potential P2/3 vector, it left
obvious room for improvement. As previously discussed (Figure 3), we elected to first synthesize the
triazine nitrile 4 analogous to an optimum dipeptide nitrile from our previous work. Figure 4 presents a
comparison of models of 4 docked into the active sites of TgCPL and HsCPL. The triazine nitrile replaces
the P1-P2 amide bond and retains the reversibly covalent nitrile interaction with the active site cysteine.
In the P2 position, we previously found that leucine is the optimal dipeptide amino acid, so in the
respective position on the triazine, which we hypothesized would project into P2, we placed an isoamyl
amine to achieve the same length into the pocket. In P3, our most active compound previously bore a 4-
fluorobenzyl, and therefore we placed the same motif in the hypothetical P3 position for the triazine
nitrile scaffold. An additional feature found both in our HTS lead and known literature analogs was the P1
morpholine. While the S1 pocket in TgCPL is absent, the morpholine in the P1 position projects into
solvent and has been shown to improve the solubility of this chemotype (Figure 4).
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Figure 4. Comparison of TgCPL and HsCPL binding sites. A) Covalent docking of 4 into TgCPL
active site and electrostatic surface mapping. Red= polar negative, White = neutral, Blue= polar
positive. (Derived from PDB: 3F75); B) Covalent docking of 4 into the active site of HsCPL (PDB:
5MAJ). Pocket view; Red= polar negative, White= neutral, Green= lipophilic
The installment of these two pendants in our first triazine analog 4 indeed provided over a 100-
fold improvement in potency over the HTS lead 3, affording an IC50 of 34 nM and 32 nM for TgCPL and
HsCPL, respectively (Table 1). These results supported our docking model, and validated our hypothesis
that we could successfully scaffold-hop from the dipeptides to the triazine, suggesting that much of our
dipeptide SAR should translate directly to the triazine series. The active site of TgCPL contains four
residues (Figure 4A) that are either non-conserved across the human cathepsin isoforms A-X, or unique
to TgCPL. Notably, the S3 pocket of TgCPL tends to be significantly more polar as compared to HsCPL,
with Gln69, Asp78, and Glu75 making up the key pocket residues. The human cathepsin L (Figure 4B) is
somewhat more lipophilic with the S3 pocket residues Glu63, Tyr72 and Leu69, respectivly. While this
presents potential for differences in ligand selectivity, the S3 pocket is shallow and somewhat
promiscuous with its preferred residues. We anticipated that the majority of our potential selectivity
would come from the S2 pocket, which tends to be deeper and somewhat more defined. Importantly, the
parasitic cathepsin bears an Asp218 in the S2 pocket, while the human isoform contains an Ala214 in the
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corresponding position. We therefore expected that selectivity for TgCPL could be achieved by exploiting
the differences in the overall topology and unique residues. As such, we chose to explore various P2 and
P3 vectors to probe these diffences and elucidate the pharmacophore for selective and potent inhibition of
TgCPL.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
SAR of P3 Vectors
Table 1. SAR of P3 vectors
O
N
N
N
R
N
N
CN
Selectivity
for
TgCPLa IC50
HsCPLb
IC50 (nM)
No.
R
(nM)
TgCPL c
0.7
HTS Lead 3
4
see Fig 1
3,500
34
2,300
32
0.9
F
F
5
6
7
47
53
20
19
33
0.4
F
0.4
F
356
0.09
F
8
53
34
0.6
F
F
F
9
48
35
62
86
35
0.7
0.6
0.8
0.2
F
10
11
12
101
110
230
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N
4
5
6
7
13
14
61,457
15,200
12
2
0.0002
0.001
8
9
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
16
17
82
39
33
27
41
0.4
0.7
0.4
N
N
107
NH2
18
230
87
0.4
H2N
H2N
19
20
21
22
23
95
137
304
32
28
296
492
6
0.3
2.2
1.6
0.2
0.4
N
Cl
O
95
36
24
107
14
1.3
25
26
64
19
22
5
0.3
0.3
27
28
29
37
17
17
41
47
3
1.1
2.8
0.2
0.1
CF3
30
47
6
O
NO2
31
32
27
42
96
14
3.6
0.3
O2N
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34
59
51
56
0.9
0.5
O2N
CN
105
7
8
9
CF3
35
36
37
740
123
970
266
91
0.4
0.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
67
0.07
a,bIC50 for Toxoplasma gondii Cathepsin L (TgCPL) or human Cathepsin L (HsCPL).
Values are mean of at least 3 independent experiments with SEM typically < 30%.
cSelectivity for Toxoplasma gondii Cathepsin L as defined by the ratio HsCPL IC50/TgCPL
IC50
We began our SAR investigation by retaining the isoamyl P2 vector and varying the P3 position
(Table 1). A scan of fluorine mono-substitution of the P3 benzyl (5 and 6) showed retention of activity,
but no significant improvement in either overall potency or selectivity. A similar outcome was seen with
the P3 di-fluoro analogs 7, 8, and 9. The non-fluorinated P3 benzyl pendant 10 exhibited roughly a 3-fold
loss of potency versus the 4-fluoro lead 4. Homologation of the linker from methyl to ethyl (11) or propyl
(12), while better tolerated for the human isoform, resulted in the same reduced potency for TgCPL. An
even greater loss of potency for the parasitic enzyme was seen when large bulky pendants like the
quinoline or naphthyl of 13 and 14 were installed. Taken together, this demonstrates that the parasitic
isoform does not productively accomodate large lipophilic vectors in the P3 position, which is consistent
with the topologically more polar nature of the S3 pocket defined by Asp78 and Glu75 (Figure 4A).
With this in mind, we tried the 2-, 3-, and 4-pyridyl pendants (15, 16, and 17) in P3, with the
objective of improving our selectivity for the parasite, as well as potentially improving our metabolic
profile by decreasing the clogP. Of these, the 3-pyridyl (16) retained potency, but unfortunately did not
offer any improvement in selectivity over the human isoform. Anilines 18, 19, and 20 were also
evaluated. We had hoped to gain a meaningful interaction with Asp78, Gln69, or Glu75 in TgCPL
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(Figure 4A), but none of the anilines were very potent for TgCPL. However, analog 20 did afford a small
measure of selectivity toward TgCPL (HsCPL IC50/TgCPL IC50 = 2.2). Analog 21 was tested to determine
if a basic amine might afford greater TgCPL selectivity, but resulted in nearly a 9-fold loss in potency
versus parent compound 4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As a comparison to our 4-fluorobenzyl pendant, we tested the 4-chlorobenzyl analog 22 to
determine the impact of a larger halogen. While equally effective against TgCPL to the 4-fluoro, this
change afforded an order of magnitude improvement in potency for HsCPL. Analog 23 was synthesized
to assess the effects of a moderately electron withdrawing acetophenone, but afforded a mild reduction in
potency against TgCPL. A methyl scan was performed to probe the space around the ring and determine if
any of the substitution patterns might lead toward some selectivity for TgCPL. While 24 and 25 did not
offer any improvement in potency, 26 was nearly twice as potent (TgCPL IC50 = 19 nM) as the parent
scaffold 4, but more strongly improved HsCPL inhibition (HsCPL IC50= 5 nM), similar to what we
observed with the 4-chloro analog 23. It is worth noting that the o-methyl benzyl analog 24, while less
potent than the 4-flurobenzyl, showed mild selectivity for TgCPL. Dimethyl analogs 27 and 28 provided
more potent inhibitors and were consistent with the trend that an ortho substitution (28) may favor
selectivity for TgCPL. Undesirably, these also increased overall molecular weight and introduced
potential metabolic hotspots, and therefore were not further pursued. The electron withdrawing 3-
trifluoromethyl 29 provided one of the more potent TgCPL inhibitors (IC50 = 17 nM), but exhibited
selectivity in favor of HsCPL. The increased MW versus 4 was also undesirable in trying to remain
within the calculated properties for BBB permeability. The corresponding 3-methoxy analog 30 was also
tested to assess the effects of electron donation into the ring; however, this did not seem to offer any
significant improvement in potency or selectivity as compared to 29. Interestingly, a nitro scan in analogs
31, 32, and 33 revealed that the o-nitrobenzyl P3 vector of 31 exhibited a 3-fold selectivity over the
human isoform (TgCPL IC50 = 27 nM, HsCPL IC50 = 96 nM), consistent with the modest selectivity we
observed with ortho-methyl analogs 24 and 28. As this was the most TgCPL-selective analog to date,
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additional ortho-substituted analogs 34, 35, 36, and 37 were synthesized in an effort to better explain the
observed selectivity. The nitrile (34), trifluoromethyl (35), and methyl ester (35) vectors were tested as
isosteres of the nitro. Unfortunately, none of these substituents reproduced the selectivity we saw with the
o-nitro vector. The o-methoxy analog 37 was also synthesized to determine if the observed effects were
due to the electron withdrawing nature of the nitro, or a conformational change due to sterics. While
tolerated in HsCPL, the potency significantly decreased against TgCPL.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Based on observations in our modeling (Figure 5), we hypothesize that the P3 pendant may have
the ability to bind outside of the S3 pocket and interact with the backbone of Asp166, giving rise to a
higher tolerance for vectors as compared to that of S2. This concept is supported by the overall lack of
robust SAR trends for the S3 pocket and the reltively high substrate tolerance in the P3 position observed
in analogs 4-47. Furthermore, a recent publication observed a similar trend in triazine nitrile inhibitors of
human cathepsin L.31
Figure 5. Potential alternative binding mode for triazine nitriles
(e.g. 4). Derived from PDB: 3F75
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SAR Analysis of P2 Vectors:
5
6
7
8
Table 2. Screen of various P2 vectors
O
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
N
N
N
CN
R
F
TgCPLa HsCPLb
Selectivity
No.
4
R
IC50
IC50
for TgCPL c
(nM)
(nM)
34
32
67
26
0.94
0.27
0.22
38
39
246
117
40
144
64
0.44
41
42
46
67
32
2
0.70
0.030
43
220
328
1.5
44
45
2,260
6,490
759
0.33
0.52
O
3,430
NH2
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8
46
47
3,590
1,410
4,090
561
1.1
NH2
NH2
9
0.40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
48
1,990
2,050
1.1
N
a,bIC50 for Toxoplasma gondii Cathepsin L (TgCPL) or human Cathepsin L (HsCPL).
Values are mean of at least 3 independent experiments with SEM typically < 30%.
cSelectivity for Toxoplasma gondii Cathepsin L as defined by the ratio HsCPL IC50/TgCPL
IC50
We then turned our attention to evaluating the SAR among the postulated S2 vectors. In general,
the papain family of proteases achieve their substrate specificity primarily from interactions in the S2
pocket.38 Based on our previous SAR studies with the dipeptide nitriles, we had determined that leucine
was the optimal amino acid for the P2 position. As already noted with 4, the isoamyl sidechain in the
respective position for the triazine nitriles afforded a huge improvement in potency to HTS lead 3 (TgCPL
IC50 = 34 nM). We wanted to first determine if the previous dipeptide SAR trends we found tracked in a
similar fashion with this new chemotype. Initially, we evaluated the effects of shorter, aliphatic vectors.
The n-propyl, n-butyl, and n-butenyl (38, 39, and 40) were tolerated in HsCPL, but all decreased in
potency for TgCPL. Consistent with our previous SAR, the shorter isobutyl S2 vector in 42, which closely
mimics a valine sidechain, slightly decreased activity for TgCPL to 67 nM, but enhanced HsCPL
inhibition (IC50 = 2 nM). Interestingly, the dehydroleucine analog 41 retained nearly equivalent potency
(TgCPL IC50 = 46 nM) to 4, a trend that was not observed in the previous dipeptide series. This indicated
to us that, while similar, the SAR between the dipeptide and triazine chemotypes was not identical and
that binding of the triazine nitrile series may be somewhat better accomodated by the enzyme. Extension
of the S2 sidechain of 4 by one carbon in 43 resulted in a significant loss of potency. We believe this may
be due to a clash in the back of the S2 pocket, consistant with the S2 size constraints observed
previously.28 Compound 44 was made to determine if the S2 pocket could tolerate a less lipophilic group
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thereby reducing overall clogP and improving compound solubility. Unfortunately, this resulted in a large
decrease in potency for both enzymes. Given that TgCPL bears a unique Asp218 versus the Ala214 in
HsCPL (Figure 4), we rationalized that inclusion of a basic group in the S2 position might afford some
selectivity, as well as improve compound solubility. As such, compounds 45- 48 were synthesized. We
had hoped that the stepwise increase in side chain length might elucidate the ideal distance required to
gain a productive interaction with Asp218. Unfortunately, these changes were not well tolerated and
resulted in a drastic drop in potency down to the low micromolar range for both enzymes, indicating that
perhaps these compounds are not binding as predicted, or that Asp218 is not oriented the way it appears
in our model.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A)
B)
O
N
O
N
Secondary
N
N
N
N
N
N
N
N
N
N
F
F
Primary
Figure 6. Predicted and actual metabolism of 4 by CYPs. A) Top-ranked metabolic
sites predicted by SmartCYP for CYP3A4. B) Metabolite ID results (incubation
with mouse liver microsomes) identifying the isoamyl S2 vector as the primary
site of oxidation.
P2 Vectors for Improved Metabolic Profile and Selectivity:
The previously developed dipeptide nitrile 2 exhibited a fair level of stability toward mouse liver
microsomes (MLM) with a half-life of t1/2 = 22 min. While the scaffold hop from dipeptide 2 to the
triazine nitrile 4 resulted in an improvement in both potency and predicted CNS profile , it also imparted
a significant drop in metabolic stability (MLM t1/2 = 7 min). The metabolic liabilities predicted by
SmartCyp (Figure 6A) led us to install the α-methyl and exchange the P1 solublizing group with 3-
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amino-oxetane in analog 50 (Table 3). Unfortunately, this did not offer any improvement to the
microsomal stability profile (MLM t1/2 = 5 min). Therefore, a metabolite ID study was performed to
experimentally determine sites of oxidative metabolism. As shown in Figure 6B oxidation of 4 occurs
predominantly on the P2 isoamyl vector and the P3 benzylic methylene. Similar oxidation of S2 isoamyl
sidechains has been reported in the development of odanacatib for the inhibition of cathepsin K, and it
was overcome by replacement of the sidechain with fluoroleucine.39 With this guidance in mind, analogs
were designed to retain the physiochemical profile of a CNS drug and simultaneously circumvent these
potential metabolic liabilities (Table 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. P2 Vectors for Improved Metabolic profile and Selectivity
O
N
N
N
R1
N
R2
N
CN
TgCPLa HsCPLb
MLM
t1/2
(min)
Selectivity
No.
4
R1
R2
IC50
IC50
for TgCPL c
(nM)
(nM)
4-Fluorobenzyl
34
26
32
15
0.94
0.58
0.25
0.61
0.72
7
4-Fluoro-α-
methylbenzyl
49
50*
51
4-Fluoro-α-
methylbenzyl
92
23
5
4-Fluorobenzyl
310
143
190
103
4-Fluoro-α-
methylbenzyl
52
4.4
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4-Fluorobenzyl
4-Fluorobenzyl
68
160
26
2.4
1.5
9
17
3.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
55
56
4-Fluorobenzyl
4-Fluorobenzyl
66,800
26
87,500
95
1.3
3.7
15
57
58
59
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
3,570
110
74
66
77
60
0.018
0.70
0.81
60
61
62
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
1,900
1,190
969
0.63
2.9
NH
326
N
H
181
1,200
6.7
>60
N
H
63
64
65
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
1,200
960
94
0.80
0.25
3.5
NH
400
O
49
170
3.5
O
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66
67
4-Fluorobenzyl
2-Nitrobenzyl
630
370
0.58
0.3
NH
3,970
1,240
8
9
N
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
F
68
4-Fluorobenzyl
1,600
517
0.33
N
H
a,bIC50 for Toxoplasma gondii Cathepsin L (TgCPL) or human Cathepsin L (HsCPL). Values are mean of at
least 3 independent experiments with SEM typically < 30%. cSelectivity for Toxoplasma gondii Cathepsin L
as defined by the ratio HsCPL IC50/TgCPL IC50. *S1 morpholine group replaced with oxetan-3-amine.
Analog 49 was prepared first to evaluate the impact of the alpha-methylbenzyl on potency since
we had previously observed a loss of potency with oxetane analog 50. In fact this single change was well
tolerated and provided a small improvement in overall potency; however, the potency improvement was
2-fold greater for HsCPL. Presumably due to the added MW and lipophilicity, compound solubility
significantly decreased versus the parent compound (thermodynamic aqueous solubility: 4 = 42 µM, 49 =
16 µM). In an effort to resolve this, we tried replacing the S1 morpholine group with oxetanamine in
analog 50. As noted previously, the potency decreased to 92 nM for TgCPL, but solubility improved to
42 µM. Analogs 51, 53, and 54 were all synthesized with the intent of blocking oxidation of the isoamyl
CH.39-40 The cyclopropyl leucine mimic (51) lost an order of magnitude in potency (TgCPL IC50 = 310
nM) compared to 4, and the t-butyl analog, 53, while 2-fold selective for TgCPL, decreased to 68 nM
against TgCPL. It should be noted, however, that these respective changes in the dipeptide nitrile series
were not tolerated nearly as well, giving us some confidence that the triazine nitrile may be a superior
scaffold with regards to potential for improving PK properties. Furthermore, this data suggests these two
chemotypes are not binding in precisely the same manner, consistent with what we had observed earlier in
our SAR. Compound 52 was synthesized to determine if the cyclopropyl and α-methylbenzyl
combination in P2 and P3 respectively would be sufficient to slow metabolism. Unfortunately, this
actually decreased the MLM t1/2 to 4.4 min, indicating a likely switch to another metabolic site, perhaps
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N-dealkylation of the P2 sidechain or oxidation of the P1-morpholine. Compound 54, bearing a
fluoroleucine in S2 (a vector also not tolerated in the dipeptide series), demonstrated excellent potency for
TgCPL (IC50 = 17 nM) and HsCPL (IC50 = 26 nM), although MLM stability was decreased to t1/2 = 3.5
min, further indicating that blocking the primary metabolic site alone is insufficient for improving
stability. The ethyl-cyclohexyl vector of 55 resulted in a significant decrease in potency for both
cathepsins, likely due to the same steric clash in the S2 pocket observed for compound 43. Interestingly,
shortening the pendant by one carbon to the methyl-cyclohexyl (56) both retained potency for TgCPL
(IC50 = 26 nM) and achieved nearly 4-fold selectivity for the parasite isoform over human. Furthermore,
it doubled the MLM t1/2 of lead 4 to 15 min. The P2 cyclopentyl in 59 is comparable to the cyclohexyl in
terms of potency, but not as selective. The smaller ring sizes in analogs 57 and 58 significantly decreased
potency for TgCPL and were not further pursued.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Given the favorable TgCPL selectivity imparted by the P2 cyclohexyl ring for 56, we also
explored incorporation of a basic amine with the objective of enhancing this selectivity through a
favorable ionic interaction with Asp218. As such, S2 piperidinylmethyl compounds 62 and 63 were
synthesized. Interestingly, the 4-piperidine of 63 resulted in sharply reduced activity against both
enzymes, while the 3-piperidine 62 demonstrated good selectivity (7-fold) for TgCPL. Despite a decrease
in overall potency to 0.180 µM, 62 exhibited a much improved MLM stability with t1/2 > 60 minutes. The
drastic improvement in stability seen for compound 62 is likely due to the added charge of the basic
amine impeding CYP binding. The addition of an extra carbon in the sidechain for analog 60 decreased
potency to 1.9 µM for TgCPL, consistent with the poor activity of the homologated cyclohexane analog
55. The ethyl 4-piperidine P2 vector 61 showed a slightly improved potency as compared to analog 60,
however it was still significantly lower than parent analog 56. The respective tetrahydropyran analogs 64
and 65 were made to determine if the basic amine was important for this observed selectivity. These
displayed a similar potency and selectivity profile, with the heteroatom being better tolerated at the 3-
position (65, TgCPL IC50 = 49 nM) than the 4-position (64, TgCPL IC50 = 400 nM). Unfortunately,
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compound 65 was not stable to MLM. Reduction of the piperidine ring size to a pyrrolidine in analog 66
abolished the selectivity and potency profile. Finally, we attempted to combine our most selective P2 (3-
piperidinylmethyl) and P3 (o-nitro benzyl) vectors with analog 67. Unfortunately, these effects were not
synergistic and resulted in a loss of both selectivity and potency, strongly suggesting that the two
congeners are not binding the same way.
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Pharmacokinetics:
Compound 4 was evaluated in a mouse PK study to benchmark the CNS profile of the triazine
nitrile chemotype and compare it to that of the dipeptide nitrile 2 (Table 4). We were pleased to find that
4 does in fact access the CNS with a brain AUC/plasma AUC = 0.61. However, the compound levels
decreased rather quickly, likely due to a combination of high volume of distribution and metabolic
instability. Nonetheless, this was a promising result indicating that the triazine nitrile scaffold has the
physiochemical profile needed to cross the blood-brain barrier.
Table 4. Pharmacokinetic studies in mice*
[Plasma]
(ng/mL)
[Brain]
(ng/g)
AUCBrain AUCPlasma
(ng*hr/g) (ng*hr/mL) AUCPlasma
AUCBrain/
No.
0.5
2
4
7
0.5
2
4
7
2
4
110
141
138
17.3
10.2
7.4
4.7
-
3.1
11.4
14.3
53.6
90.8
671
-
-
-
-
-
53.6
119
868
152
376
195
1660
1010
0.14
0.61
0.52
0.15
16.1
82.5
32
50 1,530 57.7
62 473 200
12.5
16.6
13.8
12.4
65.9
36.1
* Compounds were administered as a single IP dose at 10 mg/kg.
We hoped that by increasing the metabolic stability of the scaffold, we might gain a better
pharmacokinetic profile. We attempted to evaluate compound 56, as this analog exhibited an improved
half-life in vitro, good potency, and demonstrated some selectivity for TgCPL, but unfortunately its poor
solubility precluded in vivo dosing. As mentioned above, the replacement of the P1 morpholine of 49 with
a 3-amino-oxetane in compound 50 offered an improvement in solubility (16 µM to 42 µM respectively).
We decided to perform a PK study on 50 to determine if the additional hydrogen bond donor of the
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oxetanamine would impede our CNS penetrance, or if this could be a viable strategy to improve the
solubility of future compounds (Table 4). We were very pleased to find that in addition to retaining CNS
access, the overall exposure levels were significantly increased compared to 4. Given that the 3-piperidine
P2 vector in 62 significantly improved the metabolic stability, we also evaluated its pharmacokinetic
profile. While the exposure levels were improved in plasma versus 4, this compound had markedly
reduced BBB penetrance. Basic amines are often associated with efflux by Pgp, so the difluoro-3-
piperidine compound 68 was synthesized with the intent of attenuating the basicity of this sidechain
(Table 3). Unfortunately, this change was not well tolerated and potency was reduced to low micromolar
for this compound (TgCPL IC50 = 1.6 µM).
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Because of their markedly divergent brain:plasma ratios observed in the PK studies, compounds
50 and 62 were assessed in vitro to determine whether they are potential substrates of P-glycoprotein (P-
gp). Evaluation in an MDR1/MDCK permeability assay indicated that compound 50 has high passive
permeability and an efflux ratio <1, and thus does not appear to be a Pgp substrate (Table 5). This would
be expected to translate to good CNS penetrance, which is in agreement with our PK results. 62 also
demonstrated high passive permeability, but has an efflux ratio of over 17, indicating this compound is
likely a P-gp substrate, explaining its poor brain exposure in vivo. As noted above, this is very likely to be
due to the presence of the basic amine in 62.
Table 5. MDR1/MDCK Assay results
a
Papp
Efflux Ratio
(B-A)/(A-B)
Sample
(cm/sec)
50 (A-B)
50 (B-A)
62 (A-B)
62 (B-A)
3.55 x 10-5
7.92 x 10-6
2.59 x 10-5
4.47 x 10-4
0.223
17.2
a Values shown are the average of two sample measurements of the acceptor wells
from three replicate assays.
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In vitro efficacy studies:
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Based on their potencies vs TgCPL, five compounds were selected for evaluation of their effects
on plaque formation in a T. gondii bradyzoite cyst qPCR/plaque viability assay in vitro (Figure 7). This
assay involves isolating bradyzoites after treatment and quantifying viability by applying them to a new
monolayer of host cells. Viable bradyzoites invade the monolayer, differentiate to tachyzoites, and form a
visible plaque after 12 days of growth. Viability is calculated as the number of plaques/1000 genomes of
input bradyzoites (measure by qPCR) and expressed as a percentage of solvent (DMSO) treated
bradyzoite cysts. Positive controls in the assay included a TgCPL knockout strain of T. gondii (cpl), as
well as the irreversible TgCPL inhibitor LHVS (1)25. We were pleased to find that the triazine nitriles 4
and 50 exhibited strong efficacy at 5 M that was equivalent to the genetic knockout and irreversible
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inhibitor LHVS at 1 M, with zero plaques formed in any biological replicate. To our knowledge, this is
the first example of a reversible inhibitor of TgCPL achieving in vitro efficacy against the chronic
(bradyzoite) stage of T. gondii. Triazine nitrile analogs 56, 65, and 62, on the other hand, showed
variable efficacy. 56 and 65 were slightly less efficacious than 4 and 50 at the concentration tested,
reducing cyst viability by ~80% across three biological replicates. Only one biological replicate of
compound 62 reduced viability (<20%), suggesting that it was comparatively ineffective. Dipeptide nitrile
lead (2) demonstrated only modest activity against the bradyzoite cysts, exemplifying the improved
potential of the triazine nitrile as a therapeutic.
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Figure 7. Efficacy of triazine nitrile compounds for treatment
of in vitro bradyzoite cysts within human foreskin fibroblast
(HFF) host cells. Cultures were treated with 1 µM LHVS (1)
or 5 µM nitrile compounds daily for 7 days.
To confirm that the observed reduction in bradyzoite viability was not due to human foreskin
fibroblast (HFF) host cell viability being indirectly compromised, MTS assays were also conducted to
assess cell cytotoxicity (CC). The relatively high CC50 values indicate that none of the compounds would
be expected to cause significant host cell cytotoxicity at the concentration used in the qPCR/plaque
bradyzoite cyst viability assays (5 μM).
Table 6. MTS assay results in HFF cells.
Compound
CC50 (μM) a
65.19 (±16.69)
29.05 (±2.6)
>100
61.05 (±2.08)
34.58 ±1.33)
4
50
56
62
65
aAll values are the mean of at least three
independent experiments.
CONCLUSIONS AND PERSPECTIVES
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Throughout this project, significant advances have been made in developing small molecule
inhibitors of TgCPL. We have elucidated key SAR for the inhibition of TgCPL, developed potent and
CNS penetrant inhibitors, and demonstrated efficacy against bradyzoite cysts in vitro. We started by
conducting a high throughput screen to identify new chemotypes capable of the inhibition of Toxoplasma
gondii cathepsin L as potential new therapeutics for toxoplasmosis. The low µM triazine nitrile scaffold
(3) was selected based on the previously established literature precedent of this chemotype for inhibition
of human cathepsins, its low molecular weight, and physical properties consistent with CNS-active
compounds. Translation of the pharmacophore previously developed with our dipeptide nitrile series to
the triazine nitrile scaffold resulted in an immediate 100-fold gain in potency (TgCPL IC50 = 34 nM), but
reduced metabolic stability relative to the corresponding dipeptide. A TgCPL covalent docking model was
developed using the crystal structure of TgCPL and the crystal structure(s) of human cathepsin (HsCPL)
in complex with various dipeptide and triazine nitrile inhibitors. Using this we were able to identify key
distinctions between the parasite and human isoforms of cathepsin L. In particular, the smaller S2 pocket
and the four unique residues (Asp218, Glu75, Asp78, and Gln69) were targeted with the goal of gaining
selectivity for TgCPL. 68 analogs in this series were synthesized with varying S2 and S3 vectors to
optimize metabolic stability, CNS permeability, and selectivity over human isoforms. In general, we
found that variations in the P3 position had little impact on potency or selectivity. As expected, the bulk
of the potency and selectivity seems to be imparted by efficient binding in the S2 position. The S2 pocket
across the human cathepsins tends to be more uniformly lipophilic than TgCPL, and we predicted that
inclusion of a basic residue in the P2 position might interact favorably with non-conserved Asp218 in
TgCPL. It is worth noting that out of the nine analogs bearing an amine in P2, all exhibited IC50s for
HsCPL of >0.5 µM, supporting that the human isoform does not tolerate a basic amine in S2. TgCPL,
conversely, appears to be able to better accommodate either a nitrogen or oxygen in S2, likely due to the
polar Asp218 residue in the back of the S2 pocket, and this may represent an avenue for further TgCPL
selectivity enhancement. Work in our laboratory is underway to exploit these features and further explore
the SAR trends with various basic and polar groups in P2.
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Overall, we improved potency against TgCPL to as low as 5 nM and identified features that can
be exploited to gain selectivity vs HsCPL (up to 5-7 fold), and significantly improved metabolic stability
(up to t1/2 >60 min in MLM). Importantly, we were able to demonstrate significant brain penetration with
several analogs in vivo in mice. Although our most Tg-selective analog 62 bearing a basic amine in the P2
pendant proved to be highly susceptible to efflux in MDR1-MDCK cells in vitro and demonstrated low
brain exposure in vivo, an analog lacking this basic amine (50) was shown not to be a P-gp substrate and
achieved good exposure in the brains of mice after IP dosing, suggesting the triazine nitrile chemotype
has promise as an in vivo probe for chronic T. gondii infection studies. Synthesis of new analogs to
further improve PK and isoform selectivity are underway. Finally, we demonstrated for the first time that
in vitro treatment of T. gondii bradyzoite cysts with non-peptidic triazine nitrile inhibitors reduces
parasite viability with efficacy equivalent to a TgCPL genetic knockout, in contrast to one of our
previously developed dipeptide nitriles, highlighting the increased therapeutic potential of the triazine
template. This is the first example of a CNS penetrant, reversibly covalent TgCPL inhibitor showing
efficacy in an in vitro model of bradyzoite stage parasites. Current studies are underway to advance the
triazine nitrile series into an in vivo model of latent Toxoplasma infection.
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METHODS
Chemistry General Information: All reagents were used without further purification as received from
commercial sources unless noted otherwise. 1H NMR spectra were taken in DMSO-d6, MeOD, or CDCl3
at room temperature on Varian Inova 400 MHz or Varian Inova 500 MHz instruments. Reported chemical
shifts for the 1H NMR and 13C NMR spectra were recorded in parts per million (ppm) on the δ scale from
an internal standard of residual tetramethylsilane (0 ppm). Mass spectrometry data were obtained on either
a Micromass LCT or Agilent Q-TOF. An Agilent 1100 series HPLC with an Agilent Zorbax Eclipse
Plus−C18 column was used to determine purity of biologically tested compounds. Unless otherwise noted,
all tested compounds were determined to be >95% pure using a 6 minute gradient of 10-90% acetonitrile
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