Discovery of Selective Toxoplasma gondii Dihydrofolate Reductase Inhibitors for the Treatment of Toxoplasmosis

Article abstract of DOI:10.1021/acs.jmedchem.8b01754

A safer treatment for toxoplasmosis would be achieved by improving the selectivity and potency of dihydrofolate reductase (DHFR) inhibitors, such as pyrimethamine (1), for Toxoplasma gondii DHFR (TgDHFR) relative to human DHFR (hDHFR). We previously reported on the identification of meta-biphenyl analog 2, designed by in silico modeling of key differences in the binding pocket between TgDHFR and hDHFR. Compound 2 improves TgDHFR selectivity 6.6-fold and potency 16-fold relative to 1. Here, we report on the optimization and structure-activity relationships of this arylpiperazine series leading to the discovery of 5-(4-(3-(2-methoxypyrimidin-5-yl)phenyl)piperazin-1-yl)pyrimidine-2,4-diamine 3. Compound 3 has a TgDHFR IC₅₀ of 1.57 ± 0.11 nM and a hDHFR to TgDHFR selectivity ratio of 196, making it 89-fold more potent and 16-fold more selective than 1. Compound 3 was highly effective in control of acute infection by highly virulent strains of T. gondii in the murine model, and it possesses the best combination of selectivity, potency, and prerequisite drug-like properties to advance into IND-enabling, preclinical development.

Full text of DOI:10.1021/acs.jmedchem.8b01754

Subscriber access provided by Iowa State University | Library
Article
Discovery of Selective Toxoplasma gondii Dihydrofolate
Reductase Inhibitors for the Treatment of Toxoplasmosis
Allen Hopper, Adam Brockman, Andy Wise, Julie Gould, Jennifer Barks,
Joshua B Radke, L. David Sibley, Yongmao Zou, and Stephen Thomas
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01754 • Publication Date (Web): 09 Jan 2019
Downloaded from http://pubs.acs.org on January 10, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of Selective Toxoplasma gondii Dihydrofolate Reductase Inhibitors for the
Treatment of Toxoplasmosis
,
a
a
b
b
c
Allen T. Hopper* , Adam Brockman , Andy Wise , Julie Gould , Jennifer Barks , Joshua B.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
c
d
a
Radke , L. David Sibley , Yongmao Zou , and Stephen Thomas
a
th
Vyera Pharmaceuticals, LLC, 600 Third Avenue, 10 Floor, New York, NY 10016
bEvotec (UK) LTD, Alderley Park, Cheshire, UK SK104TG
c
Department of Molecular Microbiology, Washington University School of Medicine, 660 S.
Euclid Ave., St. Louis, MO 63130
d
th
WuXi AppTec (Tianjin) Co., Ltd. 168 NanHai Road, 10 Avenue, TEDA, Tianjin (300457)
P.R. China
ABSTRACT
A safer treatment for toxoplasmosis would be achieved by improving the selectivity and
potency of dihydrofolate reductase (DHFR) inhibitors, such as pyrimethamine (1), for
Toxoplasma gondii DHFR (TgDHFR) relative to human DHFR (hDHFR). We previously
reported on the identification of meta-biphenyl analog 2, designed by in silico modeling of key
differences in the binding pocket between TgDHFR and hDHFR. Compound 2 improves
TgDHFR selectivity 6.6-fold and potency 16-fold relative to 1. Here, we report on the
optimization and structure-activity relationships of this arylpiperazine series leading to the
discovery of 5-(4-(3-(2-methoxypyrimidin-5-yl)phenyl)piperazin-1-yl)pyrimidine-2,4-diamine 3.
Compound 3 has a TgDHFR IC of 1.57 ± 0.11 nM and a hDHFR to TgDHFR selectivity ratio
5
0
of 196, making it 89-fold more potent and 16-fold more selective than 1. Compound 3 was
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
highly effective in control of acute infection by highly virulent strains of T. gondii in the murine
model and it possesses the best combination of selectivity, potency and prerequisite drug-like
properties to advance into IND-enabling, pre-clinical development.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii (T. gondii), an
obligate intracellular parasite capable of infecting a wide range of hosts and many different types
1
of cells . Approximately one third of the population worldwide is chronically infected with
2
various strains of T. gondii . Infections occur through consuming contaminated food or water,
mother-to-child (congenital/vertical) and iatrogenic (transplanted organs and blood transfusion)
3
transmission . The parasitic infection is kept in check by the immune system where it exists in a
latent bradyzoite form contained within tissue cysts that are commonly found in skeletal muscle
4
and the central nervous system (CNS) . However, in cases where the immune system is
compromised, such as in a developing fetus or in patients with HIV, undergoing cancer
chemotherapy or immunosuppressive treatment for organ transplantation, the parasite can
5
transition to an active, fast replicating, and tissue damaging tachyzoite form . Depending upon
localization of the tachyzoites, active infection can cause, among other conditions, myocarditis,
blindness and encephalitis; and is associated with a high mortality rate for HIV patients, even for
5
-8
those on active anti-retroviral treatment .
The recommended first-line treatment for toxoplasmosis is a combination therapy based
on pyrimethamine (1) and sulfadiazine, supplemented with leucovorin (also known as folinic
8
acid) to protect against bone marrow suppression . Pyrimethamine and sulfadiazine act
synergistically on the folate metabolic pathway thereby inhibiting T. gondii proliferation and
2
ACS Paragon Plus Environment

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
survival9-10. The drugs inhibit DHFR and dihydropteroate synthase (DHPS), respectively, and
consequently block the synthesis of tetrahydrofolate, a key cofactor for thymidylate synthase.
Thymidylate synthase is a methyl transferase that produces deoxythymidine monophosphate
from deoxyuridine monophosphate. In many organisms including T. gondii and humans, this
pathway is the only means by which thymidine can be supplied, making it essential for DNA
synthesis and cellular proliferation10-11. Importantly, although the combination of pyrimethamine
and sulfadiazine can control actively proliferating forms such as tachyzoites, these agents have
little effect on the semi-dormant bradyzoite stages within tissue cysts and hence do not cure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
infection .
Adverse events associated with pyrimethamine-based therapy in toxoplasmosis often
1
3
result in a need to reduce dosing or discontinue therapy . The adverse events are mainly
mechanism-based related to inhibition of folic acid metabolism in host tissues with high
1
4
metabolic activity (e.g., epithelium and bone marrow) . In mice, pyrimethamine has only about
1
5
a 3-fold safety multiple between minimal effective dose and maximum tolerated dose .
Leucovorin, a form of tetrahydrofolate that is selectively taken up by human cells, is often co-
administered to help alleviate the impact of mechanism-based toxicity to the host16-19. The use of
pyrimethamine is also not suitable during the first trimester of pregnancy owing to its inhibition
1
9
of human DHFR which can impact fetal organogenesis . In addition, about 3% of the general
population and 30% of patients with HIV/AIDS have a hypersensitivity reaction to the
sulfonamide component of the treatment regimen20-22. Therefore, finding a standalone treatment
would be of significant benefit to the HIV patient population most at risk of toxoplasmosis
encephalitis and sulfa hypersensitivities. Finally, high doses of pyrimethamine can induce
2
3
seizures, a likely consequence of off-target pharmacology . The adverse events associated with
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pyrimethamine and sulfadiazine therapy could be reduced or eliminated by selectively inhibiting
only the parasite DHFR. There have been numerous attempts to discover more selective
inhibitors of TgDHFR, but with limited success24-28. Recently, the crystal structure of TgDHFR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
was solved . This finding has enabled comparative modeling between the human and T. gondii
enzymes, leading to the design and discovery of more selective TgDHFR small molecules such
3
0
as TRC-19 (2) .
Ideally, a next generation TgDHFR inhibitor would have sufficient selectivity so that
maximal parasiticidal activity would occur without concomitant inhibition of human hDHFR.
Therefore, we hypothesized it would be important that the compound exhibit TgDHFR to
hDHFR selectivity of at least 150-fold, which is about 10-fold greater than that of
pyrimethamine and possess low clearance to minimize maximal plasma concentrations while
achieving a long duration of maximally effective exposure. Given the parasites ability to invade
the CNS, it is also necessary that the compound readily penetrate the blood-brain barrier. Finally,
improving TgDHFR potency can reduce effective dose levels and decrease the likelihood of
compound related off-target pharmacology and associated side effects and toxicity.
In this study, we report on the SAR and lead optimization strategy underlying the
discovery of 5-(4-(3-(2-methoxypyrimidin-5-yl)phenyl)piperazin-1-yl)pyrimidine-2,4-diamine 3,
a selective and potent TgDHFR inhibitor with properties consistent with those outlined above
supporting the first standalone treatment for toxoplasmosis.
RESULTS AND DISCUSSION
4
ACS Paragon Plus Environment

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Pyrimethamine selectivity and potency. Pyrimethamine 1 is reported to be 7.6-fold
more selective for TgDHFR compared to hDHFR with a hDHFR IC of 760 ± 130 nM for
5
0
3
1
inhibiting conversion of dihydrofolate to tetrahydrofolate . More recently, the Ki for hDHFR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
32
was reported to be 470 nM . In our hands, 1 has an IC for inhibiting TgDHFR of 139 ± 49 nM
5
0
(Figure 1), which makes it 12-fold more potent for inhibiting TgDHFR over hDHFR. Although
this level of selectivity is sufficient to make 1 a preferred DHFR inhibitor for the treatment of
toxoplasmosis, as can be seen in Figure 1, it is not sufficient to preclude inhibition of hDHFR
3
3
and associated dose limiting mechanism-based toxicity . The concentration of 1 that gives
significant inhibition of TgDHFR, i.e., > IC , begins to also inhibit hDHFR by 5 to 35%. Based
80
on these data, it seemed reasonable that at least an order of magnitude and preferably 20-fold
increase in selectivity in a new compound would be sufficient to overcome the mechanism-based
toxicity issues observed with 1 and possibly enable a standalone therapy.
Early profiling data for lead 2 and comparison to DHFR inhibitor reference
3
0
standard drugs. The structure of lead 2 and DHFR inhibitor reference compounds
pyrimethamine (1), trimethoprim (4), methotrexate (5) and trimetrexate (6) are shown in Figure
2
. The DHFR inhibitory activity and selectivity, kinetic solubility, human liver microsomal
intrinsic clearance and permeability and efflux across an MDR1-MDCK cell monolayer for lead
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Figure 1 . Dose response curve of 1 for inhibition of T. gondii DHFR and human DHFR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aThe shaded blue area represents the concentration of 1 with >80% inhibition of TgDHFR and 5
to 35% hDHFR.
2 and reference compounds 1, 4, 5 and 6 are shown in Table 1. Lead 2 has a TgDHFR IC of
5
0
8
.76 ± 1.0 nM with 79-fold selectivity relative to the human enzyme. This is 6.6-fold more
selective than 1. Although this improvement in selectivity was encouraging, in our view is
insufficient to achieve a stand-alone treatment. Trimethoprim 4, sometimes used off label in
combination with sulfamethoxazole for the treatment of toxoplasmosis, was about 240-fold less
potent against TgDHFR than 1 with an IC of 33,100 nM and selectivity index of 15.
5
0
Methotrexate 5 was far more potent than 1, 4 or 2 for inhibiting hDHFR with an IC of 4.74 nM.
5
0
Interestingly, 5 has the opposite selectivity profile as compared to 1, where it is 17-fold less
potent for inhibiting TgDHFR (IC of 78.3 nM), giving a selectivity ratio of 0.061.
5
0
Trimetrexate 6, which structurally is a combination of the diaminopteridine head of 5 and
trimethoxybenzyl tail of 4, is nearly equally active at TgDHFR and hDHFR with respective
IC ’s of 1.35 and 4.07 nM, giving a selectivity index of 3.0.
5
0
6
ACS Paragon Plus Environment

Page 7 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 2. Reference compounds Pyrimethamine (1), Trimethoprim (4), Methotrexate (5),
Trimetrexate (6) and initial lead TRC-19 (2).
H N
2
N
NH2
O
OH
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H N
2
N
NH2
N
NH2
N
N
H
N
N
N
OH
N
N
O
Cl
O
O
H2N
O
1
4
5
H N
2
N
NH2
N
O
N
O
NH2
N
N
N
N
O
H
H N
2
6
2
Further characterization of 1 and 2 was performed to benchmark and guide a lead
optimization strategy (Table 1). Lead 2 has low kinetic and thermodynamic solubility (< 4 µM),
high clearance in human liver microsomes (HLM) (i.e. > liver blood flow), and low to moderate
MDCK-MDR1 permeability, all areas for improvement during lead optimization. Interestingly,
1
, which was discovered and developed in the 1940s, has near ideal CNS drug properties in
terms of solubility, HLM stability and MDR1-MDCK permeability. Properties for 1 and 2 are
summarized in Table 2, with the main differences being an increase in both MW and log P for 2,
which likely contributes to its undesirable solubility, metabolic stability and permeability profile.
Based on these data, lead optimization was initially focused on understanding the SAR
underlying selectivity and potency, while simultaneously designing analogs with lower cLog P
and higher calculated solubility in an effort to improve physical chemical properties.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Evaluation of Comparator Compounds on DHFR Inhibition, Kinetic Solubility,
Clearance in Human Liver Microsomes and MDR1-MDCK Permeability
Compound TgDHFRa
hDHFRa
Selectivity Solubilityc
HLM
MDR1-
IC (nM)
IC (nM)
Indexb
12
(µM)
184
3.9
(mL/min/kg) MDCKd
50
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
4
139 ± 14
1680 ± 230
689 ± 39
<8.6
28.4
<8.6
36 (0.83)
2.7 (0.62)
7.5 (3.5)
8.76 ± 1.0
79
33,100 ± 3400 503,000 ±
1,000
15
--
5
5
6
78.3 ± 6.7
4.74 ± 0.36
4.07 ± 0.11
0.061
3.0
--
--
--
--
--
--
1.35 ± 0.07
a Average of at least 3 independent replicates ± SEM. b Selectivity index (SI) is the hDHFR
IC /TgDHFR IC , determined within the same experiment. c Kinetic solubility. d A:B (papp
5
0
50
-6
1
0 cm/sec) (ER), where ER is efflux ratio defined as permeability in A:B/B:A directions.
a
Table 2 . Properties of 1 and 2
Compound
MW
248.7
346.4
cLog P
3.00
Log Pb
2.71
TPSA
76.8
cSol (µM)
135
1
2
3.74
3.38
83.2
3.09
a Calculated properties were performed using ChemDraw® Professional software version
17.0.0.206 (121). b Measured LogP was performed at pH 11.0 in octanol/buffer.
Structure-activity relationships for analogs of 2. Structure activity relationship (SAR)
data based on 2 for inhibition of TgDHFR and selectivity relative to hDHFR are shown in Tables
3
- 5. Initial optimization of 2 focused on a systematic approach to gain a better understanding
of its SAR with emphasis on improving DHFR selectivity (Table 3). As a basis for comparison,
8
ACS Paragon Plus Environment

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
unsubstituted analog 7 was prepared and found to lose potency (IC 209 nM) but maintained a
5
0
similar selectivity index (59-fold) relative to initial lead 2. Ortho-phenyl 8 and para-phenyl 9
were both about 4-fold less potent (IC ~ 30 nM) than 2 but differed in their selectivity profiles.
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ortho-phenyl 8 maintained similar selectivity as 2 at 58-fold, whereas para-phenyl 9 had reduced
selectivity at 11-fold. These data are consistent with modeling predictions of the binding
3
0
cavity . Replacing the distal meta-phenyl of 2 with an ortho-methyl 10 or ortho-chloro 13
resulted in complete loss of activity at the highest doses tested. This result is surprising
considering ortho-phenyl analog 8 had a TgDHFR IC of 31 nM. Meta-methyl and meta-chloro
5
0
analogs 11 and 14, respectively, were more potent and selective than corresponding para-
substituted compounds 12 and 15, supporting the hypothesis based on modeling that meta-
substituents would be preferred. Encouragingly, meta-methyl 11 while 8-fold less potent than 2,
maintained equivalent selectivity of 79-fold. Based on these findings, design focused on meta-
substituents to further probe this apparently important pocket for both selectivity and potency.
Meta-methoxy 16, meta-trifluoromethyl 17, meta-trifluoromethoxy 18 and meta-cyclopropyl 19
all had reduced potency and selectivity as compared to meta-phenyl lead 2. Adding a methyl or
ethyl to the 6-position of the diaminopyrimidine group, giving 20 and 21 respectively,
maintained or slightly improved potency over 2, but with a slight loss in selectivity.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Structure-Activity Relationships of phenylpiperazine analogs on inhibition of TgDHFR
and hDHFR
H N
2
N
NH2
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
R1
N
R2
Compound
R2
R1
TgDHFRa
hDHFRa
Selectivity
IC (nM)
IC (nM)
Indexb
5
0
50
2
7
8
9
3-phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8.76 ± 1.0
209 ± 28
30.9 ± 2.2
31.5 ± 2.7
>10,000
689 ± 39
12,500 ± 1200
1790 ± 190
347 ± 23
79
59
2-phenyl
4-phenyl
2-Me
3-Me
4-Me
2-Cl
58
11
1
1
1
1
1
1
0
1
2
3
4
5
>30,000
Not available
75.3 ± 5.5
181 ± 14
>10,000
5980 ± 320
3260 ± 110
>30,000
79
18
Not available
3-Cl
39.8 ± 5.3
45.9 ± 7.3
57.8 ± 3.4
62.1 ± 5.2
12.9 ± 1.1
29.0 ± 1.7
1540 ± 170
1090 ± 140
2710 ± 150
541 ± 43
39
24
47
8.7
63
56
4-Cl
16
3-MeO
3-CF3
1
1
7
8
3-CF O
819 ± 36
3
19
3-cyPropyl
1630 ± 45
10
ACS Paragon Plus Environment

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
2
0
1
3-phenyl
3-phenyl
Me
Et
9.19 ± 0.94
3.91 ± 0.61
237 ± 21
200 ± 16
26
51
a Average of at least 3 independent replicates ± SEM. b Selectivity index (SI) is the hDHFR
IC /TgDHFR IC .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
Structure-activity relationships for heteroaryl analogs of meta-biphenyl 2. We then
turned our focus toward improving the solubility and metabolic stability issues observed with
early lead 2 by preparing a variety of heteroaryl analogs (Table 4). We hypothesized that the
distal phenyl ring of 2 was the likely site of metabolism and that introducing nitrogen molecules
into this ring would block metabolism and increase solubility. The 2-, 3- and 4-pyridine analogs
2
2, 23 and 24 maintained or slightly improved potency and selectivity, but without much
improvement in solubility. The potency of 3-pyridine analog 23 is noteworthy at 2.7 nM, about
.3-fold more potent than 2, and with a selectivity index of 112-fold. Addition of a second
3
nitrogen into this ring to give pyrimidines, pyridazines and pyrazines provided analogs with
variable improvements in potency, selectivity, solubility and HLM metabolic stability. 5-
Pyrimidine analog 26 stood out with a 307-fold TgDHFR selectivity index and a TgDHFR IC50
of 5.2 nM, as well as markedly improved solubility (>200 uM) and metabolic stability (Clint <
8.6 mL/min/kg). The improvement in selectivity observed for 26 is nearly 4-times that of early
lead 2 and 25-fold better than 1. 2-Pyrazine 27 and 4-pyridazine 28 had promising potency with
IC s of 10 and 6.0 nM respectively, and selectivity indexes of 129 and 185-fold respectively, but
5
0
both were hampered with low kinetic solubility, precluding additional investigation with these
chemotypes. In general, heteroaryl substitutions increase TgDHFR selectivity and maintain or
slightly improve potency relative to 2. The two-fold improvement in selectivity observed for 5-
pyrimidine analog 26 compared to the 3-pyridyl analog 23 may result from one of the nitrogen
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
atoms on the distal pyrimidine ring being forced to make an unfavorable interaction in hDHFR.
The 3-pyridyl analog 23, which can rotate the nitrogen away from such an unfavorable
interaction is about 5-fold more potent at hDHFR than pyrimidine 26 (hDHFR IC 1430 nM).
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Evaluation of heteroaryl replacements of the distal phenyl of 2 on DHFR inhibition,
selectivity, solubility, human liver microsomal clearance and MDCK-MDR1 permeability.
H N
2
N
NH2
N
N
N
R2
Compound
R2
TgDHFRa
hDHFRa
Selectivityb Solubilityc
(µM)
HLMd
MDR1-
IC50 (nM)
IC50 (nM)
(CLint)
MDCKe
22
2-pyridine
3-pyridine
4-pyridine
10.3 ± 1.6 1360 ± 210
132
112
108
47.6
301
129
185
12
--
17.7
--
--
2
2
2
2
2
2
3
4
5
6
7
8
2.68 ± 0.12
4.34 ± 0.43
300 ± 55
468 ± 32
1300 ± 51
--
2.2
180
>200
1.6
5.7
18.7
<8.6
<8.6
<8.6
<8.6
17.4 (0.86)
31.7 (0.80)
20.2 (1.2)
19.3 (0.77)
6.40 (3.8)
2-pyrimidine 27.3 ± 2.3
5-pyrimidine 4.75 ± 0.34 1430 ± 100
2-pyrazine 10.3 ± 0.39 1330 ± 50
4-pyridazine 6.00 ± 0.70 1100 ± 130
a Average of at least 3 independent replicates ± SEM. b Selectivity index (SI) is the hDHFR
IC /TgDHFR IC , determined within the same experiment. c Kinetic solubility. d HLM human
50
50
-6
liver microsomes, CLint intrinsic clearance (mL/min/kg), e A:B permeability (papp 10 cm/sec),
where ER is efflux ratio defined as permeability in A:B/B:A directions.
12
ACS Paragon Plus Environment

Page 13 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
SAR for the distal 5-pyrimidine ring of 26. Next, we evaluated if selectivity could be
further improved by adding substitutions onto the 2-position of the distal 5-pyrimidine ring of 26
(Table 5). Based on the crystal structure and modeling (see Figures 3 and 4), it appeared that
substitutions at this position were not likely to have steric effects as they pointed into solvent
space. However, if the pyrimidine nitrogen atoms were making a favorable hydrogen bond
interaction with TgDHFR His-27 and an unfavorable interaction with hDHFR Pro-26, then
electron donating substituents would be predicted to enhance potency and selectivity. Methyl
analog 29 maintained TgDHFR potency (IC 3.95 nM) with only a slight reduction in selectivity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
(237-fold) relative to unsubstituted 5-pyrimidine 26. Methyl analog 29 had good solubility,
HLM stability and MDCK-MDR1 permeability. An electron donating 2-methoxy substituent
gave 3, with excellent TgDHFR potency with an IC of 1.57 nM, but a slightly reduced
5
0
selectivity index of 196. Trifluoromethyl 30 and cyclopropyl 31 were significantly less potent
against TgDHFR, less selective and had low kinetic solubility. The loss in selectivity with these
analogs is principally driven more by loss of activity against TgDHFR rather than by gaining
potency at hDHFR.
Further characterization of 3 demonstrated good kinetic solubility of 186 µM at pH 7.4,
low human microsomal clearance (<8.6 mL/min/kg) and high permeability in MDR1-MDCK
-6
cells (20.1 x 10 cm/s) with a low efflux ratio of 1.1 (Table 5). In the CEREP 44 safety screen at
a 10 µM dose level, 3 demonstrated good selectivity against all 44 targets tested, except for
3
hERG where [ H] Dofetilide binding was inhibited by 60%.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Evaluation of substitutions on the distal pyrimidine ring of 26 on DHFR inhibition,
selectivity, solubility, human liver microsomal clearance and MDCK-MDR-1 permeability
H N
2
N
NH2
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
N
R3
R3
TgDHFRa
hDHFRa
HLMd
Compound
Selectivity Solubility
MDR1-
MDCKe
19.3 (1.2)
20.1 (1.1)
--
IC (nM)
IC (nM)
indexb
237
(µM)c
150
186
17
(CLint)
<8.6
5
0
50
29
3
Me
3.95 ± 0.26
1.57 ± 0.42
32.7 ± 3.3
935 ± 46
308 ± 71
MeO
CF3
196
<8.6
3
0
2800 ± 470
973 ± 120
85.6
98
10.5
31
cypropyl 9.92 ± 0.83
12
17.4
--
a Average of at least 3 independent replicates ± SEM. b Selectivity index (SI) is the hDHFR
IC /TgDHFR IC , determined within the same experiment. c Kinetic solubility. d HLM human
5
0
50
-6
liver microsomes, CLint intrinsic clearance (mL/min/kg), e A:B permeability (papp 10 cm/sec),
where ER is efflux ratio defined as permeability in A:B/B:A directions.
Chemistry. The compounds presented here were synthesized as shown in Schemes 1
and 2. In Scheme 1, substituted piperazines (36a-n) were condensed with 5-bromouracil 33 in
the presence of KF and heating in DMSO to give the corresponding uracil derivatives 37a-n.
Chlorination using POCl and DIPEA provided intermediates 38a-n, followed by amination
3
reaction using NH /EtOH at 145 °C to give final products 2, 7-18, 20, 21 and 32. 6-Methyl and
3
14
ACS Paragon Plus Environment

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6
-ethyl diaminopyrimidine targets 20 and 21 were synthesized as described above, but starting
with the corresponding 6-alkyl-5-bromouracils 34 and 35 and condensing with biphenyl-
piperazine 36a. Piperazine intermediates 36a-n are generally commercially available or can be
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
prepared by various literature methods34-35
.
a
Scheme 1 . Synthesis of Compounds 2, 7-18, 20, 21 and 32
H
N
H
N
O
O
N
O
O
HN
a
N
HN
+
HN
Br
R2
R1
N
R1
1
3
6a-n
37a-n
3
3 R = H
R2
1
3
4 R = Me
1
35 R = Et
H N
2
N
NH2
Cl
N
Cl
c
N
b
N
N
N
R1
N
R1
N
R2
, 7-18, 20, 21 and 32
38a-n
R2
2
2
R = (a) 3-phenyl, (b) hydrogen, (c) 2-phenyl, (d) 4-phenyl, (e) 2-methyl, (f) 3-methyl, (g) 4-
methyl, (h) 2-chloro, (i) 3-chloro, (j) 4-chloro, (k) 3-methoxy, (l) 3-trifluoromethyl, (m) 3-
trifluoromethoxy, (n) 3-bromo
a Reagents and conditions: (a) KF, DMSO, 110 °C, 12 h; (b) POCl , DIPEA, 110 °C, 5 h; (c)
3
NH , EtOH, 145 °C, 24 h
3
Analogs in Scheme 2 were prepared by either Suzuki coupling of 3-bromophenyl 32 with
the corresponding boronic acids 39o-w to give targets 3, 19, 23, 24, 26 and 28-31, or Stille
2
coupling of 3-bromophenyl 32 with Sn(Bu) R 40x-z to generate compounds 22, 25 and 27.
3
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 2 . Synthesis of Compounds 3, 19, 22-31
H N
2
N
NH2
N
H N
2
N
NH2
N
N
N
a or b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
32
3
, 19, 22-31
R2
Br
2
R = (o) 5-(2-methoxypyrimidine), (p) cyclopropyl, (q) 3-pyridine, (r) 4-pyridine, (s) 5-
pyrimidine, (t) 4-pyridazine, (u) 5-(2-methylpyrimidine), (v) 5-(2-trifluoromethylpyrimidine),
(w) 5-(2-cyclopropylpyrimidine), (x) 2-pyridine, (y) 2-pyrimidine and (z) 2-pyrazine.
2
a Reagents and conditions: (a) Pd(PPh ) , R -B(OH) (39o-w), Cs CO , Dioxane/H O, 110 °C;
3
4
2
2
3
2
2
or (b) Pd (dba) , Sn(nBu) -R (40x-z), Xphos, dioxane, 100 °C
2
3
3
Figures 3 and 4. Crystal Structure of TgDHFR with pyrimidines 29 and 3 bound.
Crystal structure data was visualized and modeled using MOE software from Chemical
Consulting Group.
16
ACS Paragon Plus Environment

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Crystal structure of TgDHFR. To aide in the understanding of the observed selectivity
and guide future lead optimization efforts, several X-ray crystal structures with lead compounds
bound to TgDHFR were solved, including the complex of 5-pyrimidines 3 and 29 with TgDHFR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figures 3 and 4). Although at a high-level these data are consistent with modeling predictions,
the crystal structure provides more subtle clues into why the distal pyrimidine moiety enhances
selectivity. The phenyl groups of 3 and 29 occupy the TgDHFR cavity created near F91 and G22
(Figure 4), while the residues present in hDHFR at these positions appear to be less
accommodating. These TgDHFR phenylalanine residues (F32 and F91) create a more enclosed
hydrophobic area around the phenyl ring connected to the piperazine. This configuration also
positions the distal heteroaromatic ring into the glycine site, possibly enabling a hydrogen
bonding interaction with His 27 in the T. gondii enzyme (Figure 3). The presence of a proline
residue (Pro26) in hDHFR, at the analogous position of His 27 in TgDHFR, provides a
hypothesis for the observed subtle improvement in selectivity with the 5-pyrimidines. The
TgDHFR His27 may enable a favorable H-bond with a pyrimidine N, whereas this would not be
feasible in the human enzyme. In addition, there could be an unfavorable interaction in the
hDHFR between the pyrimidine nitrogen atoms of 3 and Pro 26. This interaction might help
explain why 3-pyridine 23 is less selective than pyrimidine 3, as the pyridine N could rotate
away from the proposed unfavorable interaction with Pro26 in the human enzyme. We
hypothesize these are the likely reasons for the observed TgDHFR selectivity. However, it is
also noted that, N64 in hDHFR blocks - interactions. Interactions at these residues are well
3
6
supported by the literature and may also contribute to the observed TgDHFR selectivity . A
search of UniProt shows that there are no reported human polymorphisms related to these amino
3
7
acids that would alter selectivity .
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Parasiticidal and anti-proliferative effects of 5-methoxypyrimidine 3 in cell-based
models. Optimized pyrimidine 3 and comparator drug 1 were evaluated in cell-based models to
investigate translation of the respective DHFR inhibitory activities into parasiticidal and
antiproliferative host cell effects. Lead 3 had a parasiticidal EC of 13 nM and 1 had an EC of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
6
80 nM against the type I RH strain of T. gondii. The 52-fold improvement in potency of 3 over
in this assay is consistent with its 89-fold potency advantage for inhibiting TgDHFR.
1
Antiproliferative effects of these compounds in human MCF-7 cells, measured using the MTT
assay, were consistent with the human DHFR data, wherein 1 and 3 had EC s of 11,600 nM and
5
0
7
,300 nM, respectively. The selectivity of both 1 (17-fold) and 3 (560-fold) in the cell-based
assays (Table 6) is in line with their DHFR selectivity profiles of 12 and 200-fold, respectively.
These data are important as they demonstrate that 3 has anti-parasiticidal activity with similar
efficacy as 1, but with substantially better potency and selectivity.
Table 6 Parasiticidal activity for 1 and 3 in T. gondii infected human foreskin fibroblast cells
as compared to the antiproliferative effects of 1 and 3 in MCF-7 cells
T. gondii Cell-Based
MCF-7
Selectivity
Indexc
Compound
a
b
EC (nM)
EC (nM)
5
0
50
3
1
13 ± 3.8
7300
560
17
680 ± 210
11,700
a EC =half maximal response concentration, average of at least 3 experiments (± SD). b MCF-
5
0
7
cell viability determined using the MTT assay in triplicate (n = 1). c Selectivity index
calculated as the MCF-7 EC /Tg cell-based EC .
50
50
18
ACS Paragon Plus Environment

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 5. Parasiticidal dose response curves of 1 and 3 in T. gondii infected human foreskin
fibroblast cells.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparison of potency against additional strains of Toxoplasma. Given the
enhanced potency of 3, we wanted to test it against different lineages of T. gondii.
Consequently, we chose a series of low passage isolates that are representative of the major
genotypes of T. gondii seen in human infection from different regions of the world. As we have
previously described, the population structure of T. gondii is comprised of 6 major clades, each
3
8
of which contains several related lineages . The majority of human cases of toxoplasmosis in
Europe and North America are due to type 2 strains39-41, and we typically use a laboratory strain
called ME49 as representative for such strains. The ME49 strain was originally isolated from a
42
sheep in the United States , and it reflects the genotype of many strains found in food animals
and which cause human toxoplasmosis. The CTG strain was originally isolated from a cat in the
United States and it represents a common genotype found in animals, although it is relatively
43
rare in humans . Type 1 strains are also reasonably abundant in North America, and they are of
interest as they are more pathogenic in many hosts including immunocompromised humans41, 44
Although most lab studies use the type 1 RH strain, use of the GT1 strain is preferable as it
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
45
preserves the entire lifecycle . The GT1 strain was originally isolated from a goat in North
4
6
America , although it is genetically similar to a number of human isolates including the RH
strain. In contrast to North America and Europe, strains in South America are dominated by
highly pathogenic lines such as types 5 (reference strain RUB) and 10 (reference strain
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
8
VAND) . The VAND and RUB strains were isolated from severe human cases of
toxoplasmosis that are characteristic of the Amazon region47-48. These strains have also been
4
9
used as reference strains for the Toxoplasma comparative genomes paper .
To monitor the effects of new antiparasitic compounds on different strains of T. gondii we
generated fire fly luciferase (FLUC) expressing lines so that we could use a common luciferase-
based growth assay to monitor growth inhibition. We compared the potency of 3 for its ability to
inhibit the growth of T. gondii strains in vitro. As shown in Table 7, the potency of 3 was highly
similar across these different lineages, with only 2 to 4-fold differences being observed in
potency. The FLUC lines used here also carry a pyrimethamine resistance allele of DHFR that
was used to generate the transgenic lines. This resistant DHFR allele confers highly level
5
0
resistance to pyrimethamine as described previously , but interestingly this did not lead to cross-
resistance to 3.
20
ACS Paragon Plus Environment

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Table 7 Growth effects of 3 on different strains of T. gondii
Geographic
Host of origin
Type
EC50 (nM)
Strain
Distribution
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
GT1
goat
North America
1
2
18.0 ± 9.3
8.3 ± 2.9
ME49
sheep
North America
Europe
CTG
RUB
Cat
North America
South America
South America
3
5
29.8 ± 15.4
7.6 ± 3.3
human
human
VAND
10
11.2 ± 0.5
a Average of at least 3 independent replicates ± SEM
Mouse pharmacokinetic and brain to plasma exposure studies. 2-Methoxypyrimidine
was selected over 2-methylpyrimidine 29 and unsubstituted pyrimidine 26 for further in vivo
3
characterization owing to its substantially improved stability in mouse liver microsomes (MLM).
Preclinical models of toxoplasmosis are primarily performed in mice and therefore a compound
with sufficient oral bioavailability, and plasma and CNS exposure to support murine efficacy
testing was desired. Fortunately, 3 had moderate mouse liver microsome (MLM) intrinsic
clearance (Clint) of 56.3 mL/min/Kg, which is 63% of mouse liver blood flow (LBF), whereas
2
9 and 26 both had high predicted clearance with MLM Clint values of 431 and 2,700
mL/min/Kg, respectively, multiples above mouse LBF. The oral and IV PK parameters in
mouse are summarized in Table 8. Compound 3 has low to moderate clearance of 10.6
mL/min/kg, a volume of distribution of 1.14 L/kg and a half-life of 3.9 h after a 1.0 mg/kg, iv
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dose. Oral bioavailability after a 0.83 mg/kg, po dose was 47.3% with a Cmax of 178 ng/mL 30
minutes after dosing. The unbound fraction (%) in mouse plasma is 8.7 ± 0.2 and in brain
homogenate 2.4 ± 0.3, determined using an equilibrium dialysis method. Compound 3 was freely
permeable into the mouse CNS at 0.5 hours after a 10 mg/kg oral dose, the concentration in brain
was 2,560 ± 240 ng/g and in blood 1,610 ± 580 ng/mL giving a brain to blood ratio of 1.7 ± 0.6.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 8 Pharmacokinetics of compound 3 in mouse
Dose
Cmax, po Tmax, po AUC0-last, po
CL, iv
Vd, iv
t
½, iv
%
F
Route
(
mg/kg)
(ng/mL)
--
(h)
--
(ng•h/mL) (mL/min/kg) (L/kg) (h)
IV
1.0
--
--
10.6
--
1.14
--
3.9
--
PO
0.83
47.3
178
0.05
750
a Oral bioavailability (%F), area under the concentration-time curve from time zero to the last
measurable concentration (AUC0-last), clearance (CL), maximum observed plasma concentration
(
Cmax), intravenous (IV), per os (PO), elimination half-life (t ), time to Cmax (Tmax), volume of
½
distribution (Vd)
In vivo efficacy of 1 and 3 in a murine model of acute Toxoplasmosis. Compounds 1 and 3
were tested independently in a murine model of acute toxoplasmosis and observed to improve
survival in a dose-dependent manner (Figures 6a and 6b). Female CD-1 mice were inoculated
intraperitoneally (IP) with 3,000 tachyzoites of the highly virulent RH strain of T. gondii on
Day 0. In parallel, 3 negative control mice were inoculated with vehicle, lacking the parasite.
The parasite strain used in this study was genetically modified to constitutively express the
®
51
enzyme luciferase and enable infection monitoring via an in vivo imaging system (IVIS ) .
Mice were monitored for survival for 30 days with intermittent IVIS monitoring (data not
22
ACS Paragon Plus Environment

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
shown). On day 4 of dosing, compound exposure was assessed by micro-sampling from tail vein
from 3 mice per group at 1, 4 and 12 h post-dose and 3 mice per group at 2, 8 and 24 h post-dose
(data shown in Figures 7a and 7b).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6a.a Kaplan-Meier survival curves of compound 1 in CD-1 female mice (n = 12 for
drug treated groups, n = 9 for vehicle group) infected with 3,000 RH type 1 T. gondii tachyzoites
on day 0 followed by oral administration of 1 beginning on day 1 through day 7.
a
Mice were carefully monitored for signs of infection, including by intermittently imaging (data
not shown).
Compound 3 at doses of 10 mg/kg, QD or BID for 7 days, yielded 100% survival for 30
days (end of study), while doses of 1 and 3 mg/kg produced similar results on survival as
comparator compound 1 did at 18 mg/kg BID and 54 mg/kg QD, respectively. Survival data for
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
in this study were consistent with published literature52-54. All mice treated with vehicle died
within 6 to 7 days. It should be noted that the single animals that were lost in the 3 mg/kg
compound 3 group and 54 mg/kg compound 1 group were believed to be due to handling errors
during imaging rather than active T. gondii infection as no signs of infection or bioluminescence
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(data not shown) were observed for these animals during the study. Consistent with the
improved in vitro potency of 3 relative to 1, it was also more potent in vivo producing similar
efficacy at doses about 18-fold lower.
Figure 6b.a Kaplan-Meier survival curves of compound 3 in CD-1 female mice (n = 12 for
drug treated groups, n = 9 for vehicle group) infected with 3,000 RH type 1 T. gondii tachyzoites
on day 0 followed by oral administration of 3 beginning on day 1 through day 7.
a
Mice were carefully monitored for signs of infection, including by intermittently imaging (data
not shown).
24
ACS Paragon Plus Environment

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Cure rates were assessed by subinfection of naive mice with combined homogenized brain
and lung tissue from mice that survived 30 days. The tissues from two surviving mice of the
same dosing group were injected into one donor mouse. Infection with a single T. gondii
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45
parasite of the type I RH strain is believed to be 100% lethal in naive mice ; therefore, sub-
inoculated naive mice that survived for 15 days and were without signs of infection, was
indicative of a cure (i.e., the donor mice were devoid of parasite). It should be noted that the RH
strain of T. gondii does not typically form tissue cysts. Therefore, a cure in this model may not
translate to a cure with clinically relevant strains that form tissue cysts. Nonetheless, it is clear
both 1 and 3 are strongly efficacious in eradicating the tissue damaging and life threating
tachyzoite form of the parasite.
Compound 3 had a cure rate of 50% in the 1 and 3 mg/kg groups and 100% in the 10
mg/kg groups. Pyrimethamine 1 was 100% curative at the 54 mg/kg dose and 33% to 67%
curative at the 18 mg/kg BID dose. These data suggest that inhibition of TgDHFR is sufficient
for maximal efficacy as a stand-alone therapy (e.g., without coadministration of a DHPS
inhibitor). Regarding the results for pyrimethamine 1, the human equivalent dose of 54 mg/kg in
mouse (approximately 300 mg/day in a 70-kg individual) cannot be administered in patients
owing to dose-limiting, mechanism-based safety issues.
For both compounds, the onset of effect on survival time occurred at exposures near or
slightly above the T. gondii cell-based EC values (1.0 mg/kg for 3 and 18 mg/kg for 1), with
5
0
maximal effects occurring at dose levels of 3 mg/kg and 10 mg/kg for 3 and 54 mg/kg for 1
Figures 7a and 7b), which exceed the cell-based EC s.
(
5
0
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 7a. Day 4 [plasma]free exposure of 1 from the acute murine model of toxoplasmosis
plotted as free plasma (nM) levels versus time.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7b. Day 4 [plasma]free exposure of 3 from the acute murine model of toxoplasmosis
plotted as free plasma (nM) levels versus time.
26
ACS Paragon Plus Environment

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CONCLUSIONS
Rational drug design led to the identification of several highly selective and potent
arylpiperazine-based TgDHFR inhibitors. The structure-activity relationship for these
compounds provides proof-of-concept for the feasibility of discovering a next generation
toxoplasmosis treatment with improved potency, selectivity, and safety. 2-Methoxypyrimidine 3,
with 16-fold better selectivity for TgDHFR and 89-fold better potency as compared to
pyrimethamine 1, and desirable DMPK and safety properties, was selected for exploration in
preclinical models of toxoplasmosis. Compound 3 was effective in radical cure of acute infection
with the highly virulent type I RH strain in the murine model and showed similar potency across
multiple parasite lineages in cell-based parasite growth assays. Based on the efficacy observed,
we hypothesize it will be feasible to achieve parasiticidal levels of 3 in patients that are below
concentrations that would inhibit hDHFR, thus providing a safer and more efficacious therapy
than is currently available and possibly obviating the need for sulfonamide combination therapy.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Materials and Methods.
General. All synthetic chemistry, DMPK, T. gondii DHFR crystal structures with 3 and 29, and
DHFR in vitro pharmacology was performed at WuXi App Tec at their China facilities in Tianjin
and Shanghai. Work with Toxoplasma gondii was performed at Evotec in Manchester, UK and
in Prof. Sibley’s lab at Washington University, St. Louis. Proton NMR spectra were recorded on
a Varian 400 MHz NMR. LCMS were taken on a quadrupole Mass Spectrometer on Shimadzu
LCMS 2010 (Column: sepax ODS 50×2.0 mm, 5 µm) or Agilent 1200 HPLC, 1956 MSD
(Column: Shim-pack XR-ODS 30×3.0 mm，2.2 µm) operating in ES (+) ionization mode. All
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
final compounds were greater than 95% pure based on HPLC UV% AUC. Structures were
1
determined by H NMR and LC/MS. Final compounds in this manuscript are not known to
interfere with the assays here in (i.e., they are not PAINS).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All animal studies were conducted at WuXi App Tec in China and Evotec in Manchester, UK
and were performed in accordance with the institutional guidelines for these Countries and
Laboratories.
LC/MS Methods
Method A: Run on a Shimadzu LC-20AB with a MS 2010 detector using a Luna-C18(1) column
(2.0*30mm, 3um) at 40 C. Mobile phase A was 0.037% (v/v) aqueous TFA and mobile phase
B was 0.018% (v/v) TFA in acetonitrile. The flow rate was 0.8 mL/min from 0.01 to 1.51 min,
then 1.2 mL/min from 1.52 to 2.00 min. The gradient ran from 90% mobile phase A to 10%
mobile phase A over 1.15 min then remained at 10% mobile phase A through 1.65 min then back
to 90% mobile phase A at 1.66 min and was maintained at 90% mobile phase A through 2.0 min.
The UV detection was 220 nm and the MS was measured in positive ion mode.
Method B: Run on an Agilent 1200 with a MS 6120 detector using an Xbridge Shield RP18
column (2.1*50mm, 5um) at 40 C. Mobile phase A was 10 mM aqueous NH HCO and mobile
4
3
phase B was acetonitrile. The flow rate was 1.0 mL/min from 0.01 to 2.48 min, then 1.2 mL/min
from 2.50 to 3.00 min. The gradient ran from 90% mobile phase A to 20% mobile phase A over
2
.00 min then remained at 20% mobile phase A through 2.48 min then back to 90% mobile
phase A at 2.50 min and maintained at 90% mobile phase A through 3.0 min. The UV detection
was 220 nm and the MS was measured in positive ion mode.
28
ACS Paragon Plus Environment

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Method C: Run on an Agilent 1200 with a MS 6120 detector using an Xbridge Shield RP18
column (2.1*50mm, 5um) at 40 C. Mobile phase A was 10 mM aqueous NH HCO and mobile
4
3
phase B was acetonitrile. The flow rate was 1.0 mL/min from 0.01 to 2.50 min, then 1.2 mL/min
from 2.51 to 3.00 min. The gradient ran from 70% mobile phase A to 10% mobile phase A over
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
.50 min then remained at 10% mobile phase A through 2.50 min then back to 70% mobile
phase A at 2.51 min and maintained at 70% mobile phase A through 3.0 min. The UV detection
was 220 nm and the MS was measured in positive ion mode.
Method D: Run on an Agilent 1200 with a MS 6120 detector using a Venusil XBP-C18 column
(2.1*50mm, 5um) at 40 C. Mobile phase A was 0.0375% aqueous TFA and mobile phase B
was 0.018% TFA in acetonitrile. The flow rate was 0.8 mL/min from 0.01 to 4.5 min. The
gradient was maintained at 99% mobile phase A from 0.00 min to 0.40 min, then the gradient ran
from 99% mobile phase A to 10% mobile phase A over 3.00 min then to 0% mobile phase A
over 0.45 min; then back to 99% mobile phase A over 0.01 min and maintained here for 0.55
min The UV detection was 220 nm and the MS was measured in positive ion mode.
Chemistry
The compounds in Figure 2, were sourced as follows. Compound 2 was prepared as described
3
0
1
previously . Pyrimethamine (1) was sourced from the drug manufacturer and H NMR and
LC/MS data confirmed structure and purity of 99.9%. Trimethoprim (4) was purchased from a
1
commercial supplier as the lactate salt and H NMR and LC/MS data confirmed structure with a
1
purity of 98.7%. Methotrexate (5) was purchased from a commercial supplier and H NMR and
LC/MS data confirmed structure with a purity near 100%. Trimetrexate (6) was purchased from
29
ACS Paragon Plus Environment