Development of a series of aryl pyrimidine kynurenine monooxygenase inhibitors as potential therapeutic agents for the treatment of Huntingtons disease

Article abstract of DOI:10.1021/jm501350y

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

Full text of DOI:10.1021/jm501350y

Article
pubs.acs.org/jmc
Development of a Series of Aryl Pyrimidine Kynurenine
Monooxygenase Inhibitors as Potential Therapeutic Agents for the
Treatment of Huntington’s Disease
Leticia M. Toledo-Sherman,*,§ Michael E. Prime,† Ladislav Mrzljak,¶ Maria G. Beconi,§ Alan Beresford,▽
Frederick A. Brookfield,† Christopher J. Brown,† Isabell Cardaun,‡ Stephen M. Courtney,†
Ulrike Dijkman,⊥ Estelle Hamelin-Flegg,† Peter D. Johnson,† Valerie Kempf,# Kathy Lyons,∥
Kimberly Matthews,▽ William L. Mitchell,† Catherine O’Connell,▽ Paula Pena,† Kendall Powell,#
Arash Rassoulpour,⊥ Laura Reed,† Wolfgang Reindl,‡ Suganathan Selvaratnam,† Weslyn Ward Friley,#
Derek A. Weddell,† Naomi E. Went,† Patricia Wheelan,# Christin Winkler,‡ Dirk Winkler,‡ John Wityak,§
Christopher J. Yarnold,† Dawn Yates,▽ Ignacio Munoz-Sanjuan,§ and Celia Dominguez§
†Evotec (U.K.) Ltd, 114 Innovation Drive, Milton Park, Abingdon, OX14 4RZ, U.K.
‡Evotec AG, Manfred Eigen Campus, Essener Bogen 7, 22419 Hamburg, Germany
§CHDI Management/CHDI Foundation, 6080 Center Drive, Suite 100, Los Angeles, California 90405, United States
∥Kathryn A. Lyons, P.O. Box 64, Holland, New York 14080, United States
⊥Brains On-Line LLC, 7000 Shoreline Court, South San Francisco, California 94080, United States
#Tandem Laboratories, 2202 Ellis Road, Durham, North Carolina 27703, United States
▽Charles River Laboratories, Chesterford Research Park, Saffron Walden, Essex, U.K.
¶CHDI Management/CHDI Foundation, 155 Village Boulevard, Suite 200, Princeton, New Jersey 08540, United States
S
* Supporting Information
ABSTRACT: We report on the development of a series of pyrimidine carboxylic
acids that are potent and selective inhibitors of kynurenine monooxygenase and
competitive for kynurenine. We describe the SAR for this novel series and report on
their inhibition of KMO activity in biochemical and cellular assays and their
selectivity against other kynurenine pathway enzymes. We describe the
optimization process that led to the identification of a program lead compound
with a suitable ADME/PK profile for therapeutic development. We demonstrate
that systemic inhibition of KMO in vivo with this lead compound provides
pharmacodynamic evidence for modulation of kynurenine pathway metabolites
both in the periphery and in the central nervous system.
The Kynurenine Pathway. The kynurenine pathway (KP)
(Figure 1) is the major metabolic pathway in mammals to
catabolize tryptophan leading to de novo synthesis of NAD+.4
Tryptophan is catabolized to NAD+ through a series of
enzymatic reactions commencing with oxidative conversion to
N-formyl kynurenine by indolamine dioxygenase, tryptophan
dioxygenase, indolamine-pyrrole dioxygenase (IDO/TDO/
INDOL1) dependent on tissue and cellular distribution, and
each is regulated by different stimuli.5,6 Hydrolysis of the
aldehyde moiety by kynurenine formamidase forms the key
intermediate metabolite L-kynurenine (KYN). KYN is a central
substrate of three critical enzymes in the pathway, resulting in
INTRODUCTION
■
Huntington’s disease (HD) is an autosomal dominant disease
caused by a CAG repeat expansion in the huntingtin (HTT)
gene that encodes an expanded polyglutamine tract in the
mutant huntingtin (mHTT) protein. Clinical manifestations of
this disease include motor and cognitive impairment and
psychiatric disturbances, as well as metabolic abnormalities,1
with disease onset typically occurring between the ages of 30
and 50, and death within 15 to 20 years. While mHTT is
ubiquitously expressed throughout the body, striatal medium
spiny neurons display the highest susceptibility to cell death in
HD2 and account for the manifestations of motor, cognitive,
and psychiatric symptoms of this disease. There are currently
no treatments for HD that modify disease progression, and few
palliative treatments are available.3
Received: September 2, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Kynurenine pathway (KP) with neurotoxic metabolites colored red and neuroprotective metabolites colored green.
Figure 2. Structures of known KMO inhibitors and their potencies against KMO.
three different branches, kynurenine monooxygenase (KMO),
kynureninase (KYNU), and kynurenine amino transferases
(KATs). KMO hydroxylates kynurenine to 3-hydroxykynur-
enine (3-HK), followed by hydrolysis by kynureninase to yield
3-hydroxy anthranilic acid (3HAA). Additionally, KYNU
hydrolyzes KYN to yield anthranilic acid (AA), which is in
then oxidized to 3HAA by anthranilate 3-monooxygenase.
Subsequently, 3HAA serves as a substrate for anthranilate 3-
monooxygenase, which catalyzes an oxidative ring opening of
the aryl group to form 2-amino-3-carboxymuconate semi-
aldehyde, which then cyclizes to quinolinic acid (QA). QA is
converted to nicotinic acid mononucleotide by the enzyme
quinolinate phosphoribosyltransferase (QPRT) and further
processed by several enzymatic steps beyond the KP to form
NAD+.7 An orthogonal branch of the pathway proceeds from
KYN, leading to its transamination by kynurenine amino
transferases (KATs) to yield kynurenic acid (KYNA).
Deregulation of the KP has been causally implicated in the
pathophysiology of HD and other neurodegenerative diseases.
While the exact mechanisms by which mHTT affects the
normal functioning and regulation of the KP remain unclear,
there is substantial evidence from human postmortem samples
and animal models for deregulation of the pathway in the
brain.8,9 For example, early grade HD patients show increased
B
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3-HK/KYNA and QA/KYNA ratios in striatum and cortex
compared to controls.10 Elevation of 3-HK levels has also been
reported in the brain of the R6/2 transgenic HD mouse
model.11 Other HD models have been shown to have elevated
levels of 3-HK and QA. The full-length mHTT YAC128 mouse
model, a much later-onset model of the disease, shows
elevation of 3-HK in cortex, striatum, and cerebellum and
elevation of QA in striatum and cortex at 8 and 12 months of
age. Similarly, the HdhQ111 knock-in HD mouse model has
elevations of 3-HK and QA at 15 months of age.11 Evidence of
KP deregulation in HD is not limited to the brain; HD patients
have significantly higher plasma KYN levels and KYN/
tryptophan ratios than healthy controls, suggesting deregulation
or imbalance toward the QA-producing branches of the
pathway in HD patients.12 The collective evidence for the
involvement of these metabolites in the pathophysiology of HD
highlights the KP as an attractive therapeutic intervention
point.13
KYN and KYNA but also the neurotoxic metabolites 3-HK and
QA.27 The ability to modulate the KP peripherally, leading to a
central elevation of KYN and KYNA, is essential to our
therapeutic approach. Blocking KMO selectively would lead to
an elevation of KYN and KYNA without affecting (or
decreasing) the levels of 3-HK and QA in the brain, assuming
that the KMO inhibitors modulate enzymatic activity centrally
at sufficient levels to prevent 3-HK generation. Three
metabolites of the pathway have been implicated in synaptic
physiology or in the pathogenesis of HD; 3-HK and QA are
thought to be neurotoxic, and KYNA is postulated to be
neuroprotective.28,29 QA can act as an NMDA receptor agonist,
with an EC50 of 7−200 μM for NR2A- and NR2B-containing
subunits of the NMDAR.30 When exogenously administered at
high concentrations into the brain, QA can cause excitotoxic
lesions in rodents and monkeys producing motor disturbances
reminiscent of human HD due to widespread medium spiny
neuron cell death.31,32,32,33 3-HK is a known reactive radical
generator34 and can exacerbate QA-mediated lesions.35,36
KYNA, an antagonist of the NMDA receptor acting at the
glycine coagonist site at micromolar concentrations,37 has been
shown to protect against QA- and kainate-induced neuro-
toxicity.38 Conversely, administration of dopaminergic drugs
such as apomorphine (known to cause transient depression of
KYNA extracellular levels in brain), increases the size of QA-
induced lesions in rat brain.39
Several KMO inhibitors based on the structure of the natural
substrate KYN (1) have been described in the literature (Figure
2). One of the earliest published examples, m-Nitrobenzoyla-
lanine (m-NBA, 2), has an IC50 of 0.9 μM against KMO;
intraperitoneal administration (400 mg/kg) of this compound
to mice decreases central levels of 3-HK while increasing the
levels of KYNA in blood and brain.40 Further structural
manipulation of this compound produced FCE 28833A (3), a
0.2 μM inhibitor with improved efficiency at increasing the
levels of KYNA in both blood and brain.41 Removal of the
amine and enantioselective conformational restriction, via
incorporation of a cyclopropyl ring, led to the (1S,2S) isomer
UPF-648 (4) with 20 nM potency.16 Unlike 2 and 3,
compound 4 maintains excellent KMO inhibitory activity at
the cellular level. Compound 4 has also been shown to be
efficacious in protecting against striatal QA injections in mice
lacking KAT II.42 Recently this compound was found to
increase KYNA levels and be neuroprotective in a fly model of
HD.15 A structurally unrelated compound, Ro-61-8048 (5), a
37 nM inhibitor in biochemical assays, has been widely used to
study the effects of KMO inhibition in vivo.43 This compound
raises levels of KYNA in the brain and has shown protection in
a variety of animal models of neurodegenerative diseases.43−46
Based on our previous work47 (Beconi et al., 2013, data not
shown), 5 does not cross the blood-brain barrier and therefore
peripheral KMO inhibition and subsequent increase of KYN
and KYNA in the brain are thought to account for its
neuroprotective effects.
From a therapeutic point of view, KMO is located at a critical
branching point in the KP, its activity mediating opposite
effects in the levels of 3-HK and QA versus KYNA. Inhibition
of KMO should shunt the pathway away from the toxic
metabolites 3-HK and QA and toward the formation of the
protective metabolite KYNA. Evidence implicating KMO as a
potential HD treatment has been provided by a functional
genomics screen in yeast in which deletion of the yeast
homologue of KMO, BNA4, resulted in suppression of mHTT-
induced toxicity,14 and by pharmacological and genetic
inhibition of KMO in a fly model of HD that led to increased
levels of KYNA and ameliorated neurodegeneration.15 Pretreat-
ment with UPF-64816 (4, Figure 2), a 20 nM KMO inhibitor,
protects KAT II-deficient mice against QA-induced lesions.17
It is important to note that not all branches and enzymes of
the pathway are expressed in the same tissue and by the same
cells; some degree of specialization occurs, and several
kynurenines are released from cells and function as signaling
molecules in addition to their role as metabolic precursors. In
blood monocytes, for instance, the tryptophan catabolic
pathway is fully expressed, and tryptophan can be catabolized
to QA.18 In the brain, KMO is mainly expressed by microglia,19
whereas KAT II is expressed predominantly in astrocytes and
neurons.20 Therefore, pathway metabolites such as 3-HK and
KYNA are produced in different cells, as these branches of the
pathway are segregated.21,22 Furthermore, the interplay of the
KP between the peripheral (blood and other tissues) and
central compartments is not fully understood.23 While
tryptophan, KYN, and 3-HK can cross the blood−brain barrier
from the periphery via the large neutral amino acid transporter,
acid-containing metabolites such as KYNA and QA have very
limited brain penetration and are produced in situ in the
brain.24 The levels of KYNA in the brain can be influenced via
influx of KYN into the brain from the periphery;25 for example,
systemic administration of 100 mg/kg of KYN to mice has been
shown to significantly increase central levels of both KYN and
KYNA.26 But systemic administration of KYN also leads to an
elevation of the toxic metabolites 3-HK and QA in brains,
limiting the potential beneficial effects of KYN administration
for chronic indications. Similarly, in a variety of pro-
inflammatory conditions, activation of IDO leads to an increase
in KYN metabolites in the brain. Upstream activation of
tryptophan catabolism toward KYN generation via systemic
administration of IDO-activator interferon-γ increases KP
metabolite levels in the brains of treated animals, including
While assessing the structures of the known KMO inhibitors,
we designed a class of pyrimidine carboxylic acids with
remarkably good potencies against KMO (such as compounds
6 and 7, Figure 3). The aryl pyrimidine was proposed since the
pyrimidine N3 nitrogen would mimic the carbonyl oxygen of
KYN and the KYN mimics m-NBA and FCE 28833A, while the
nitrogen at the N1 position would mimic the amine nitrogen.
We report the structure−activity relationship of this novel
series of selective KMO inhibitors and describe the chemical
C
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
in good yields. Methyl derivative 20 was treated with NBS in
the presence of AIBN and the resulting bromide treated with
sodium methoxide to generate the methoxy methyl analogue
21.54 Palladium-catalyzed carbonylation of these analogues,
followed by hydrolysis, gave the desired target compounds 22−
27. Methyl ether 27 was subsequently treated with lithium
hydroxide to generate pyrimidinol 28.55
Replacement of the carboxylic acid moiety required a more
bespoke approach, and these are described in Scheme 4.
Conversion of chloropyrimidine 29 to the corresponding nitrile
30 could be achieved in good yield via palladium-catalyzed
cross-coupling with zinc cyanide.56 Reaction of nitrile 30 with
sodium azide then generated tetrazole 31 which could be
further derivatized to the 1- or 2-methyl tetrazoles (32 or 33)
using sodium hydride and methyl iodide.57 Alternatively,
reaction of nitrile 30 with hydroxylamine led to the generation
of intermediate 34 which could then be ring closed using either
carbonyl diimidazole to give oxadiazolone 35, or triethyl
orthoformate to generate oxadiazole 36.58,59
Amine 37 was generated by nucleophilic substitution of
chloropyrimidine 29 with ammonia and, in turn, could be
converted to sulfonamides 38 and 39, or amides 40 and 41,
with sodium hydride and the corresponding sulfonyl chloride
or acid chloride. Alternatively, heterocycles such as imidazole or
pyrazole could be introduced directly following deprotonation
of the heterocycle and microwave-promoted nucleophilic
substitution of chloropyrimidine 30 to afford targets such as
42, 43, or 44.60
Figure 3. Example pyrimidine carboxylic acid KMO inhibitors.
optimization of physicochemical properties, which led to
excellent ADME/PK drug properties. PK/PD relationships
have been established for several of our program compounds
and are described. We also describe the ability of our lead KMO
inhibitor to modulate in vivo peripheral and central levels of
kynurenines in wild type rats. Work describing the biological
effects of our lead molecule in mouse models of Huntington’s
disease will be described elsewhere.
CHEMISTRY
■
General methods used for the synthesis of the pyrimidine KMO
inhibitors are described in Schemes 1−4. Suitably substituted
phenyl boronic acids or boronic esters were purchased
commercially, or prepared using methods previously described
by our group.48,49 Suzuki coupling with 4,6-dichloropyrimine
led to the generation of a chloropyrimidine intermediate which
was subsequently converted to the corresponding methyl ester
via palladium-catalyzed carbonylation (Scheme 1).50,51 The
resulting esters were then hydrolyzed with sodium hydroxide to
give the desired carboxylic acids in good yields.
RESULTS AND DISCUSSION
■
We evaluated the compounds described in this report using
biochemical and cellular KMO inhibition assays that utilized
LC-MS/MS to monitor the effect of our compounds in
inhibiting the conversion of kynurenine to 3-hydroxykynur-
enine by KMO (Scheme 5). These assays have been previously
described elsewhere but are briefly mentioned here.61 We
developed biochemical assays using mitochondrial fractions
derived from mice, rats (the two HD rodent species in which in
vivo efficacy of compounds would be evaluated), and human
liver tissues. The mass spectroscopy-based biochemical assays
measure the conversion of exogenously added KYN (typically
100 μM) to 3HK.
Further manipulations to the left-hand side of the molecule
were achieved using the chemistry described in Scheme 2. In
this case, nucleophiles such as morpholine, 3,4-dichlorophenol,
or 3,4-dichloroaniline underwent SNAr displacement with 4,6-
dichloropyrimidine in good yield, and the resulting chloropyr-
imidines 8−10 were subsequently converted to the correspond-
ing methyl esters via palladium-catalyzed carbonylation.52
Alternatively, palladium-catalyzed cross-coupling of methyl 6-
chloropyrimidine-4-carboxylate with cyclohexyl zinc bromide
gave intermediate 11.53 Sodium hydroxide-mediated hydrolysis
of the methyl esters then led to the isolation of the target
carboxylic acids 12−15.
We also established cellular assays in stably transfected CHO
cell lines expressing human or rodent KMO enzymes, as well as
in primary cells with endogenous levels of KMO, to provide a
more relevant physiological context. These assays, which have
been described previously,61 also monitor the direct conversion
Manipulations of the 3- and 5-positions of the pyrimidine
ring are described in Scheme 3. Suzuki coupling of 3,4-
dichlorophenyl boronic acid with 4,6-dichloropyrimidine
substituted at the 3- or 5-positions gave compounds 16−20
a
Scheme 1. General Synthesis of Pyrimidinecarboxylic Acids
a
Reagents and conditions: (a) KOAc, bis-pinacol borane, PdCl2(dppf)2, DMSO, 80 °C, 18 h; (b) Pd(PPh3)4, K2CO3, dioxane, 90 °C, 2 h; (c) CO (5
bar), MeOH, PdCl2(dppf)2, NEt3, 50 °C, 18 h; (d) 2 M NaOH, THF, 4 h.
D
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Modification via SNAr Displacement
a
Reagents and conditions: (a) iPrOH, 80 °C, morpholine or 3,4-dichloroaniline, 12 h; (b) 3,4-dichlorophenol, tBuOK, tBuOH, 35 °C; (c) cyclohexyl
zinc bromide, PdCl2(dppf)2, THF, 45 °C, 15 h; (d) CO (5 bar), MeOH, PdCl2(dppf)2, NEt3, 50 °C, 18 h; (e) 2 M NaOH, THF, 4 h.
a
Scheme 3. Modification to the Core Pyrimidine Ring
a
Reagents and conditions: (a) K3PO4, PdCl2(dppf)2, DMF, 80 °C, 16 h; (b)NBS, AIBN, 1,2-DCE, 90 °C, 18 h; (c) NaOMe, MeOH, r.t., 20 h; (d)
CO (5 bar), MeOH, PdCl2(dppf)2, NEt3, 50 °C, 18 h; (e) 2 M NaOH, THF, 4 h; (f) LiCl, DMF, 145 °C, 18 h.
of exogenous KYN to 3HK by mass spectroscopy. We used
freshly isolated neonatal rat microglial cultures and human
peripheral blood mononuclear cells (PBMCs) stimulated to
express KMO via a pro-inflammatory induction protocol, to
provide relevant cellular contexts. These assays were employed
routinely to evaluate compound effects on cellular KMO
inhibition in a central (microglia) and a peripheral (blood cells)
tissue population. Compound toxicity was evaluated using the
ATPlite luminescence assay in the same mammalian CHO cell
system to monitor potential confounding compound effects on
cell proliferation and cytotoxicity.
As mentioned previously, our initial design drew inspiration
from the structures of KYN, m-NBA, and FCE 28833A. In our
aryl pyrimidines, the pyrimidine N3 nitrogen was thought to
mimic the carbonyl oxygen, while the nitrogen at the N1
position would mimic the amine nitrogen, although the
hydrogen-bonding nature of these heteroatoms is different
than in KYN and its mimics. Given the available SAR around
the KYN mimics and UPF-648, our first compounds were the
3-chlorophenyl and 3,4-dichlorophenyl pyrimidine acids (6 and
7). These pyrimidine acids displayed excellent KMO inhibitory
potency with IC50 values of 0.5 nM and 0.6 nM, respectively, in
the human KMO biochemical assay. While the potencies of
these two compounds were almost identical at the biochemical
level, the cellular activity of 7 was superior to that for analogue
6, most likely due to enhanced cellular penetration. An overlap
(Figure 4B) of aryl pyrimidines 6 and 7 with KYN (3) and
UPF-648 (4) (Figure 4A) shows the high fidelity of shape and a
subset of pharmacophoric features shared between these
compounds.
Our SAR focused on three distinct areas: left hand side
modifications, core pyrimidine modifications, and replacement
of the carboxylic acid moiety (Figure 5). The subnanomolar
KMO potencies of 6 and 7 focused our attention on this
chemotype, and we set out to survey the SAR of this compound
class. The first set of investigations we undertook involved
modifications of the biaryl system, removal of the chlorines,
replacement of the chlorophenyl ring with a saturated ring,
introduction of large substituents, and replacement of the
chlorophenyl with a heteroaryl ring. All modifications shown in
Table 1 resulted in detrimental effects on compound potency.
While the SAR around 4 has not been published, we surmised a
similar mode of binding and SAR around the dichlorophenyl
E
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. General Synthesis of Example Carboxylic Acid Replacements
a
Reagents and conditions: (a) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C, 18 h; (b) TMS-N3, dibutyl(oxo)stannane, PhMe, 115 °C, 24 h; (c) NaH, MeI,
DMF, 40 °C, 18 h; (d) H2N−OH, DIPEA, EtOH, 70 °C, 2 h; (e) CDI, DBU, dioxane, 110 °C, 1 h; (f) HC(OCH3)3, 100 °C, 3 h; (g) Sat. aq. NH3,
100 °C, 16 h; (h) R2Cl, NaH, dioxane, r.t., 18 h; (i) imidazole or pyrazole, EtOH, μwave, 140 °C, 20 min.; (j) imidazalone, NaH, DMF, μwave, 140
°C, 10 min; (k) NaH, MeI, DMF, 3 h.
the pyrimidine group to understand the requirements for
hydrogen binding of this core. In the limited SAR around the
KYN mimics, it appears that the amino group, which in our
structures is replaced by an aromatic nitrogen (N1 position),
does not contribute much to the binding energy. This can be
appreciated in the structure of 4 which lacks an amino
functionality and is 10-fold more active than 3. We were
therefore curious to understand the importance of the
pyrimidine nitrogen atoms and their correspondence to the
hydrogen bonding groups in 3 and 4. The 1-pyridyl analogue
49 had an IC50 of 3.2 nM, while the 3-pyridyl analogue 50,
which maintains a hydrogen bond acceptor in a position
equivalent to the ketone oxygen of 3 and 4, showed a drop in
activity with an IC50 of 90 nM (Table 2). This pointed to the
requirement of a potential hydrogen bond acceptor atom at
position 1 (N1) of the pyrimidine. Similarly, introduction of a
Scheme 5. Conversion of Kynurenine to 3-
Hydroxykynurenine by KMO
for both chemotypes, and we therefore turned our attention to
other areas of the series to further our understanding of SAR.
While the pyrimidine core resembles in shape the six-
membered hydrogen bonded ring of benzoylalanine (3, Figure
4A), the nature and positions of hydrogen bond donor and
acceptor functionality and aromaticity of the pyrimidine core is
different. We therefore turned our attention to the SAR around
Figure 4. A. Structures of FCE 28833A (3), UPF-648 (4), and pyrimidine acid (7). B. Overlap of the pyrimidine acid (7) with FCE 28833A (3) and
UPF-648 (4).
F
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Overview of SAR carried out on the pyrimidine scaffold.
a
a
Table 1. SAR around the Biaryl System
Table 2. SAR around the Pyrimidine Core
hKMO
IC50
(μM)
compd
no.
hKMO CHO
IC50 (μM)
R1
R2
X
Y
7
H
H
N
N
0.0006
0.0032
0.09
0.033
0.17
1.1
49
50
22
23
28
27
25
24
26
H
H
CH
N
N
H
H
CH
N
CH3
H
H
N
0.012
0.006
0.14
0.3
F
N
N
9.2
H
OH
N
N
3.5
H
OCH3
CH3
N
N
0.04
4.4
H
N
N
0.010
0.26
1.5
H
NH2
N
N
5.5
H
CH2OCH3
N
N
0.033
4.3
a
SD = 5−10%, IC50 values are the average of triplicate tests.
than 17-fold relative to 7, likely indicating a need for
coplanarity of the biaryl ring systems, as a methyl at position
5 poses substantial strain on the biaryl ring torsions. We also
investigated the incorporation of other small groups at the 5-
position such as fluoro (23), hydroxyl (28), methoxy (27), and
amino (24); while the loss of activity at the biochemical level
varied from 10-fold for the 5-fluoro to >400-fold for the 5-
amino, the cellular activity was in unacceptable ranges in all
cases. This data indicate very little tolerance for substitution on
the pyrimidine ring. It appears that maintaining coplanarity of
the biaryl system is important. Likewise, substitutions adjacent
to the nitrogen at position 1 of the pyrimidine were not
tolerated, likely indicating a hydrogen bond acceptor function
for this atom.
While it has been demonstrated that levels of KYNA in brain
can be increased by peripheral inhibition of KMO with
compounds that do not cross the blood−brain barrier,43 we
wanted to obtain a brain-penetrable KMO inhibitor that may
inhibit the catabolism of KYN into the presumed neurotoxic
metabolites 3-HK and QA in the brain. One common feature of
the benchmark compounds (3 and 4) and our pyrimidine
inhibitors is the presence of a carboxylic acid. It is well-known
that acids, which carry a negative charge at physiological pH, do
not readily penetrate the brain. Furthermore, exploratory PK
carried out on our pyrimidine acid (6, data not shown)
confirmed the negligible brain exposure for this class of
compounds in rodents. At the time this work was performed,
the crystal structure of KMO was not available, but review of
a
NT = not tested, SD = 5−10%, IC50 values are the average of
triplicate tests.
methyl substituent at the 2 position of the pyrimidine core (22)
resulted in a loss of activity of 20-fold over compound 7, also
reinforcing the potential role of the N1 pyrimidine as an
acceptor atom, as the close proximity of a methyl group could
pose steric hindrance on such hydrogen bonding interaction.
Pyridine 49 displayed only a 5-fold drop of activity as compared
to pyrimidine 7; however, a 5-fold loss of cellular activity was
also observed. Likewise, placement of a methyl at position 5 of
the pyrimidine core (25) results in a loss of activity of greater
G
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. A. Active site from crystal structure of UPF-648 complex with yeast KMO (pdb code: 4j36). B. Homology model of human KMO and
proposed binding model of compound 7.
structural information and sequence alignment of KMO with a
related enzyme class, the flavin-containing monooxygenases,62
indicated the presence of two critical arginine residues in the
proximity of the putative binding site that we hypothesized
would be involved in the binding of the KYN substrate and our
inhibitors. We hypothesized that the critical carboxylate group
in our compounds was likely engaged in an ionic salt bridge to a
guanidinium moiety of one of the arginines, and that such
interaction was critical for the binding of the KYN substrate to
KMO. It has been estimated that the occurrence of such
arginine guanidinium−carboxylate salt bridges account for
about 40% of the pairs of ionic group interactions found in
proteins.63 The recent publication of the yeast KMO structure
in complex with UPF-64864 (Figure 6A) confirms such critical
interaction between the carboxylate group of UPF-648 and
arginine residue Arg83 (yeast numbering).
Given the realization that a carboxylate group is incompatible
with good brain penetration, we investigated the SAR around
acid isosteres or groups with attenuated acidity with the aim of
discovering compounds with enhanced brain exposure. Table 3
summarizes a subset of these efforts and clearly shows the
requirement for an acidic group, as only acid replacements with
low pKa values were able to maintain inhibitory activity for
KMO. The tetrazole carboxylic acid isostere can exist in two
tautomeric states; the calculated pKa values for these tautomeric
forms in example 31 are 3.5 and 6.9, respectively. Similarly,
oxadiazolone 35 has calculated pKa values of 2.93 and 8.4,
respectively. Both compounds retained KMO inhibitory activity
with IC50 values of 2 and 12 nM, respectively. Likewise, acidic
acyl sulfonamide 51 (calculated pKa = 3.9) had an IC50 of 25
nM, but all other nonacidic replacements were either inactive or
displayed weak KMO activity. Further investigations of
compound 51 revealed instability issues, and this compound
was not pursued further. Given the possibility of an equilibrium
between acidic and nonacidic tautomeric states and the
possibility that the nonacidic form may display brain
penetration, we performed PK on tetrazole 31 and
oxadiazolone 35 (data not shown), but neither compound
displayed brain penetration.
continue to optimize the substitutions on the phenyl ring. We
set to further optimize activity and permeability of the
molecule, hoping to minimize the impact of the acid moiety
on the blood−brain barrier and thus allow us to obtain
adequate brain exposure. We continue to explore the effect of
functionalities at the ortho, meta, and para positions and
combinations thereof (Table 4). While the introduction of
fluorine in the ortho position was tolerated (see examples 57,
59, and 60), introduction of a larger group such as
trifluoromethyl (64) resulted in a loss of activity. A wide
range of small functional groups were tolerated at the para and
meta positions, but a clear preference was observed for
combinations where the meta position was a halogen such as
chloro or fluoro and the para position was either chloro, fluoro,
methyl, trifluoromethyl, or small alkoxy. While the m-chloro
and m-fluoro, in combinations as mentioned above, had
equivalent effects at the biochemical level, cellular potency
was best maintained with the m-chloro and small alkyl or alkoxy
para substituents. Large substituents such as 4-(2-ethoxy)-
morpholine (69, 70) at the para or meta position were less well
tolerated at the biochemical levels and rendered the
compounds inactive at the cellular level. The combinations of
chloro at the meta position with small alkoxy groups at the para
position consistently resulted in compounds with excellent
KMO inhibitory activities and cellular potencies. In the crystal
structure of UPF-648 with bacterial KMO,64 the 3,4-
dichlorophenyl group of UPF-648 sits in a tight binding site
surrounded by hydrophobic residues Pro321, Phe322, Phe246,
Leu234, Ile232, Leu221, and Met230, while a water of
crystallization hydrogen-bonded to Thr244 sits in close
proximity to the 4-chloro atom. On the basis of this
observation, we propose a similar binding mode for our
compounds, where the 3-chloro-4-alkoxyphenyl group would
be accommodated into a hydrophobic pocket made up by
hydrophobic residues Pro305, Phe306, Phe232, Leu220, Ile218,
Leu207, and Met216 and that the 4-alkoxy oxygen could
hydrogen bond to the hydroxyl moiety of Thr230 (human
numbering) also likely via a water molecule (Figure 6B).
On the basis of the favorable biochemical and cellular activity
obtained with the 3-chloro-4-alkoxyphenyl group, we expanded
the SAR around the alkoxy functionality (Table 5). Similar
biochemical and cellular potencies were attained with methoxy
Having confirmed that an acidic functionality was required
for KMO inhibitory activity, and also having tried various pro-
drug strategies (data not shown) unsuccessfully, we decided to
H
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 3. Replacement of the Carboxylic Acid
a
N/A = not applicable, NT = not tested, SD = 5−10%, IC50 values are the average of triplicate tests.
I
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 4. Structure−Activity Exploration of Phenyl Substitutions
a
NT = not tested, SD = 5−10%, IC50 values are the average of triplicate tests.
J
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 5. 3-Chlorophenyl Pyrimidine Acids: Optimization of the Para Position
a
NT = not tested, SD = 5−10%, IC50 values are the average of triplicate tests.
(73), trifluoromethoxy (72), and isopropoxy (74). Exploration
of cycloalkoxy groups at the para position produced a
consistent drop in potency with increasing ring size from the
cyclopropyl (75), to cyclobutyl (78), to cyclopentyl (80), and
to cyclohexyl (81) rings. Interestingly, incorporation of a
hydrogen-bond acceptor into the ring system restores some of
the potency; for example, oxetane (79) displays a 5-fold
increase in activity over cyclobutyl (78). Consistent with our
model of the human protein, the oxetane oxygen would be
within direct hydrogen bonding distance of Thr230. We were
surprised to note the 12-fold difference in activity between the
cyclopropyl (75) and isopropyl (74) analogues. We postulated
that the more constrained cyclic groups could be better
accommodated in the tight hydrophobic pocket. A potential π-
stacking interaction with Phe232 is likely responsible for the 0.8
nM IC50 of benzofuran (77). Extending out the cyclopropyl
ring with the introduction of a methylene unit (76) results in a
98-fold drop in activity with respect to the cyclopropoxy
analogue (75), consistent with the tightness of the pocket as
observed in the bacterial KMO structure.
Permeability in Caco-2 cells and stability in liver microsomes
were evaluated for compounds with overall favorable
biochemical and cellular potency profile. All compounds in
Table 5 were tested in human, rat, and mouse microsomes and
were found to be stable (intrinsic clearance <0.02 μg/min/mg
protein) and displayed high permeability in Caco-2 cells. We
further evaluated our compounds potential to be substrates of
the P-glycoprotein (PGP, MDR1) and the ATP-binding
cassette subfamily G member 2 (BCRP) efflux pumps. These
data are given in Table 5 as effective efflux ratios (EER) of the
permeability of compounds in cell lines overexpressing the
respective efflux pump vs the permeability on the same cell
lines without overexpression (EER MDCK-MDR1/MDCK-wt
or EER MDCK-BCRP/MDCK-wt, methods provided as
Supporting Information).65 The compounds evaluated were
not PGP substrates but were found to be substrates of BCRP.
K
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 6. KMO Inhibition in Human, Mouse, and Rat Biochemical and Cellular Assays for a Set of Leading Inhibitors
a
NT = not tested, SD = 5−10%, IC50 values are the average of triplicate tests.
In addition to the human enzymatic assay, we profiled our
compounds in assays for rat and mouse KMO (Table 6) since
there are Huntington’s disease models in these species. In
addition, we determined that our compounds were selective for
KMO over the other two kynurenine-processing enzymes in
the pathway, the KATs and KYNU.51 In agreement with the
cellular KMO potency observed in the KMO-expressing CHO
stably transfected cell lines, our compounds were very potent in
primary rat microglia and in primary human PBMCs, which
express endogenous levels of KMO (Table 6). While our
leading compounds were acids, their overall physicochemical
and ADME properties were quite good; they had good cellular
permeability in Caco-2 cells, were stable in microsomal
cultures, and were not substrates of the PGP efflux pump.
We prioritized compound 75 as a representative lead, based on
its activity in the biochemical and cellular assays, its selectivity
profile, and its in vitro ADME properties. Here we report the in
vivo pharmacokinetic and pharmacodynamics effects of
compound 75 in Sprague−Dawley rats.
Table 7. Summary of Pharmacokinetic Parameters following
a Single Acute iv (6.2 mg equiv/kg) or po (12 mg equiv/kg)
Dose of 75 to Nonfasted Male Sprague−Dawley Rats
plasma
brain
PK
IV 6.2 mg PO 12 mg IV 6.2 mg PO 12 mg
parameters
units
equiv/kg
12
equiv/kg
7.6
equiv/kg
0.230
equiv/kg
0.12
AUCN
(0−
μM·h·kg/
mg
infinity)
F
%
N/A
N/A
64
N/A
0.25
N/A
observed
maxnorm
μM·kg/mg
1.9
0.035
C
observed
Tmax
h
N/A
1.0
0.083
1.0
CL
MRT
Vdss
mL/h/kg
290
1.1
N/A
N/A
N/A
2.4
N/A
N/A
N/A
0.70
N/A
N/A
N/A
1.7
h
L/kg
h
0.32
1.8
half-life
(t1/2
)
a
N/A = not applicable due to matrix or route of administration.
PK studies were performed in nonfasted male Sprague−
Dawley rats to support pharmacodynamic studies by both iv
and po routes. Animals were dosed with a single iv dose of 75
at 6.2 mg equiv/kg formulated as a 1.24 mg equiv/mL solution
in DMSO:PEG400:water (10:40:50 v/v/v) or orally at 12 mg
equiv/kg as a 1.2 mg equiv/mL solution in 10% hydroxypropyl-
β-cyclodextrin of 75. This compound exhibited a relatively low
clearance of 291 mL/h/kg, small to moderate Vdss of 0.32 L/kg,
and a plasma half-life of 1.8 h (Table 7). Clearance rate is
consistent with in vitro metabolism studies in hepatic fractions
from cultures of various species, which indicated no or
negligible metabolism for this compound (data not shown).
Following oral administration, 75 was rapidly and well absorbed
with Cmax norm of 1.9 μM·kg/mg at 1 h, PO AUCN of 4.7 μM·
hr·kg/mg, and good oral bioavailability of 64%.
Plasma, liver, kidney, cortex, striatum, and CSF concen-
trations in nonfasted male Sprague−Dawley rats were measured
from 0.25 to 12 h (Table 8, Figure 7) after a single oral dose of
10 mg equiv/kg formulated as a 2 mg equiv/mL solution in
10% hydroxypropyl-β-cyclodextrin. Compound 75 exposure in
liver and kidney, the primary organs of elimination, were higher
than in plasma; particularly in kidney where dose normalized
area under the curve (AUCN) was >13-fold higher (70 μM·h·
kg/mg) vs plasma (5.3 μM·h·kg/mg). Consistent with the
compounds acidic nature, its distribution to brain tissues
(cortex and striatum) and CSF was limited. However, in
microdialysis studies in brain tissues (shown below) and in CSF
measurements, we could detect free-compound exposures near
L
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 8. Summary of Pharmacokinetic Parameters in Plasma, Cortex Striatum, CSF, Kidney, and Liver following an Oral Dose
(10 mg equiv/kg) of 75 to Fed Male Sprague−Dawley rats
dose regimen: 10 mg equiv/kg single po dose
a
a
PK parameters
units
plasma
CSF
cortex
striatum
kidney
liver
b
AUCN (0−inf)
μM·h·kg/mg
μM·kg/mg
5.3
2.6
0.5
1.7
0.014
0.011
0.5
0.056
0.030
0.5
0.10
0.047
0.5
70
20
C
maxNorm
26
9.3
0.5
2.5
observed Tmax
half-life (t1/2)
h
h
0.5
2.0
nd
1.9
2.3
a
b
From nonperfused brains. AUCN value for CSF was from 0 to 8 h since half-life could not be determined (N/D) due to insufficient data points to
define elimination phase.
Figure 7. Concentration vs time profiles of compound 75 following a
10 mg equiv/kg oral dose to male Sprague−Dawley rats in plasma,
kidney, liver, cortex, striatum, and CSF.
Figure 8. Brain concentration vs time profile of compound 75
following a single 5 mg equiv/kg iv dose to FVB (WT), Bcrp(−/−),
Mdr1a/b(−/−), and Mdr1a/b(−/−)/Bcrp(−/−) male mice.
the EC50 of compound 75 for rat KMO, as judged by its
potency in the rat microglia KMO assay.
To investigate whether compound 75 was being effluxed
from the rodent blood−brain barrier (which could explain its
low brain:plasma ratios), the compound was dosed to Bcrp(−/
−), Mdr1a/b(−/−) and Mdr1a/b(−/−)/Bcrp(−/−) knock
out (KO) and wild-type (WT) mice (Table 9, Figure 8,
the activity of BCRP is unlikely to account for the low level
brain distribution of this series.
Having determined that active efflux is likely not the main
contributor to limited brain exposure of compound 75, we
determined the extent of plasma-protein binding of compound
75 in rat and mouse. Compound 75 was found to be
moderately bound to plasma proteins with a 5.0% and 3.6%
unbound fraction in rat and mouse, respectively. We believe
that the limited brain exposure of compound 75 is likely a result
of poor passive permeability at the blood-brain barrier due to
the presence of the negatively charged acid moiety.
Modulation of KP metabolite levels in plasma and in the
CNS were followed as pharmacodynamics markers of target
engagement. In support of our drug discovery program, we
developed LC-MS/MS methods for the following KP
metabolites: KYN, 3HK, KYNA, AA and QA (these methods
are provided as Supporting Information). Typically, selected
compounds from the program were evaluated in rats for their
ability to modulate KYN and 3HK levels, as would be expected
for a KMO inhibitor. In addition, and because of the
importance in addressing pathway flux through other
kynurenine-processing enzymes (both in plasma and in the
brain), we simultaneously monitored the levels of KYNA and
AA, which reflect flux through KATs and Kynureninase,
respectively.
Table 9. Summary of Pharmacokinetic Parameters for 75
following a Single iv (5 mg equiv/kg) Bolus Dose to
a
Nonfasted Male Mice
BCRP(−/
−) +
WT(FVB/ BCRP(−/ MDR1a(−/ MDR1a(−/
mouse genotype
N)
3.1
−)
−)
2.6
−)
AUCN
(0−
plasma
3.6
3.6
infinity)
μM·h·
kg/mg
brain
0.066
N/A
0.17
1.2
0.073
0.83
0.23
1.2
plasma fold change
over WT
brain fold change over
WT
N/A
2.5
1.1
3.5
a
N/A: not applicable.
methods provided as Supporting Information). In vivo,
compound 75 did not appear to be a PGP substrate based
on similar AUCN in WT vs Mdr1a/b(−/−) KO mice. A
marginal increase of 2.5- to 3.5-fold in brain AUCN was
observed in both Bcrp(−/−) and Mdr1a/b(−/−)/Bcrp(−/
−)KO mice versus WT, respectively. These findings suggest
that distribution of compound 75 to brain is partially
dependent on BCRP, but not MDR1, expression, although
In an initial experiment, we monitored the pharmacodynamic
effects of compound 75 after a 10 mg/kg oral dose to freely
moving Sprague−Dawley rats. We measured KP metabolites in
plasma at multiple time points (Figure 9), and we monitored
changes in the extracellular levels of kynurenines in the
striatum, as assessed by microdialysis (Figure 10; methods are
provided as Supporting Information). Compound levels in both
M
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 9. Modulation of KP metabolites in plasma of Sprague−Dawley rats after administration of a 10 mg/kg po dose of compound 75.
plasma and brain dialysates were measured to establish a PK/
PD response. Consistent with previous observations of high
compound exposure in plasma and peripheral tissues, we
observed significant plasma elevations in KYN, KYNA and AA
and a significant decrease in basal levels of 3-HK, as expected
for a KMO inhibitor administered systemically (Figure 9).
Importantly, at this oral dose we observed a pharmacodynamic
effect which lasted for 8−12 h, depending on the kynurenine
metabolite in question.
Our PK analysis indicated that compound 75 had limited
distribution to brain tissues. However, given that in striatum we
could detect dialysate levels of compound 75 reaching the EC50
for microglial rat KMO (Figure 10B), we wanted to test
whether the compound could modulate CNS levels of
kynurenine metabolites via a central mechanism. As previously
reported, peripheral inhibition of KMO activity would lead to
an influx of KYN from blood to the brain.24,25,66,67 To evaluate
the extent of brain extracellular levels of kynurenine
metabolites, we measured compound 75 levels with quantita-
tive microdialysis using a metaquant MQ probe,68 and levels of
kynurenines using a second probe implanted in the
contralateral striatum. An AUC (0−240 min) of 66 nM*h for
75 in striatal extracellular fluid was determined. This represents
the “free” level of 75, confirming the limited brain penetration
in the rat observed in the pharmacokinetics experiments, but
also indicating compound 75 levels close to its EC50 for KMO
in rat microglia.
showed that there was a significant increase of KYN (F(12,
148) = 30.93, p < 0.001), AA over time from t = 40−240 min
(F(12, 151) = 34.12, p < 0.001) and for KYNA from t = 60−
240 min (F(12, 150) = 31.88, p < 0.001) in the striatal dialysate
level, as compared with t = 0 min. In contrast, levels of 3-HK
only rose 2-fold between t = 160−240 min (F(12, 139) = 9.98,
p < 0.001) in the extracellular compartment (Figure 10). This
minimal alteration in 3-HK levels in brain dialysate is suggestive
that perhaps we can exert some degree of KMO inhibition
centrally in rats. A complete lack of inhibition of central KMO
would be expected to yield a large elevation of 3-HK levels in
the brain, as the influx of peripheral KYN would presumably be
catabolized by microglial KMO to produce 3-HK. Indeed, we
have observed a large increase in extracellular 3-HK when KYN
is directly administered to rats (data not shown). Attempts to
measure central levels of QA, the other downstream metabolite
of KMO, were unsuccessful as our QA detection methods were
not sensitive enough given the small samples from the
microdialysis probes. However, definitive experiments would
require an assessment of compound 75 effects in the presence
of exogenous administration of tryptophan or kynurenine to
boost the low levels of 3HK detected centrally.
Given the presumed neurotoxic role of 3HK and QA in the
pathogenesis of HD, we conducted additional experiments to
evaluate whether compound 75 could, dose-dependently, also
modulate QA in vivo. In this study, we extended the initial
observations in plasma by monitoring kynurenine levels in a
multiple dose study (from 1 to 100 mg/kg po). The effects of
compound 75 on KYN, KYNA, and AA levels in plasma are
very robust and dose-dependent (see Figure 11). In contrast,
the modulation of 3-HK levels in plasma was found to be less
dose-dependent. Due to the instability of the hydroxylated
kynurenine and the sensitivity of the methods developed, the
effects of KMO inhibition on 3HK are less profound (in many
instances, levels of 3HK where at the limit of quantitation
Microdialysis experiments clearly indicate a profound
elevation in extracellular levels of KYN, AA and KYNA
centrally, as expected based on the peripheral modulation of
KYN. Whether any extent of central inhibition of KMO by
compound 75 contributed to these elevations is unclear. The
microdialysis studies revealed that we can obtain a PD effect of
4 h or longer for KYN and KYNA after a single oral dose of 10
mg/kg (Figure 11). Statistical evaluation by 2-way ANOVA
N
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 10. Pharmacodynamic effects of compound 75 in rat kynurenines as measured by a microdialysis probe implanted in the striatum. A.
Terminal plasma levels of 75 (t = 240 min post dose). B. Free drug unbound level of 75 in striatal extracellular fluid as measured by microdialysis.
C−F. Modulation of KP metabolites in striatum brain microdialysate after administration of a 10 mg/kg po dose of compound 75 to wild-type rats.
(LOQ) for the assay). In contrast to the observed effects on
3HK levels, compound 75 produced a significant dose-
dependent decrease in QA levels in plasma. These results
show the effects of compound 75 in blocking catabolism of
kynurenine downstream of KMO activity. Altogether, these
studies support the selective effect of compound 75 in
modulating KMO activity in vivo. We have shown that the
inhibition of KMO after oral dosing leads to a dose-
dependently elevation of the “upstream” kynurenines (KYN,
KYNA and AA) in plasma, and an inhibition of 3HK and QA
levels (two metabolites “downstream” of KMO).
nervous system. Oral administration of compound 75 in rats
produces the expected modulation of KP metabolites in plasma,
that is, a dose-proportional increase in KYN, KYNA, and AA
levels, and a decrease in 3-HK and QA levels. These effects are
also observed in the tissues monitored (liver and kidney). As
predicted from published studies, and given its ready transport
into the brain, the increase of KYN in plasma after compound
75 administration leads to an increase of KYN levels in brain
compartments (in tissues, in the extracellular fluid, and in
CSF). We also observe an elevation of KYN catabolic products,
KYNA and AA. As would be expected from the increase of
brain KYN and the minimal compound exposure, we see an
increase in 3-HK levels in brain. However, compared to the
magnitude of increases in KYNA and AA, the 3-HK increase is
minimal. Given that the free concentration of 75 in rat striatum
intracellular space is close to the cellular EC50 in rat microglia,
there is likely a certain degree of KMO inhibition in the rat
brain.
CONCLUSIONS
■
We have identified a series of pyrimidine carboxylic acids that
are potent inhibitors of KMO both in vitro and in vivo.
Through iterative medicinal chemistry our team has been able
to optimize this series to produce compounds that potently
inhibit KMO in both biochemical and cellular assays. We have
shown that these compounds are selective for KMO over other
KP enzymes that utilize KYN as a substrate, and demonstrate
that systemic inhibition of KMO in vivo with compound 75
provides pharmacodynamic evidence for modulation of KP
metabolites both in the periphery as well as in the central
Compound 75 and several other program leads have suitable
ADME/PK profiles for further development and will be
evaluated as potential therapeutics for Huntington’s disease.
The profile of this class of compounds (with poor brain
exposure) allows us to test the hypothesis of whether
O
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 11. Pharmacodynamic effects of compound 75 in terminal plasma of Sprague−Dawley rats. Rats were dosed with various concentrations of
compound 75 (from 3 to 100 mg/kg po), and terminal tissue/plasma levels of KYN (panel A), 3HK (panel B), KYNA (Panel C), and QA (Panels
D) were analyzed at various time points.
2788 dual wavelength UV detector. Mass spectra were obtained over
the range m/z 150 to 850 at a sampling rate of two scans per second
using Waters LCT or analytical HPLC-MS on Shimadzu LCMS-
2010EV systems using reverse phase Water Atlantis dC18 columns (3
μm, 2.1 × 100 mm), gradient 5−100% B (A = water/0.1% formic acid,
B = acetonitrile/0.1% formic acid) over 7 min, injection volume 3 μL,
flow = 0.6 mL/min. UV spectra were recorded at 215 nm using a
Waters 2996 photodiode array. Data were integrated and reported
using Shimadzu psiport software. All compounds display purity of
>95% as determined by this method, unless stated otherwise. Accurate
mass measurements were carried out using a Waters Micromass LCT
Premier orthogonal acceleration time-of-flight mass spectrometer 4
GHz TDC with LockSpray enable.
General Procedure A. 6-(3,4-Dichlorophenyl)pyrimidine-4-car-
boxylic Acid (7). Potassium carbonate (2 M solution, 40.0 mL, 94.3
mmol) was added in one portion to a stirred solution of 3,4-
dichlorophenyl boronic acid (4.5 g, 23.6 mmol) and 4,6-dichloropyr-
imidine (5.0 g, 37.0 mmol) in dioxane (80 mL). The mixture was
degassed with nitrogen for 5 min, after which time Pd(PPh3)4 (1.1 g,
0.9 mmol) was added in one portion, and the mixture was then heated
to 90 °C and stirred at this temperature for 2 h under a nitrogen
atmosphere. After this time, the reaction mixture was cooled to room
temperature and concentrated. The resulting residue was suspended in
DCM (300 mL) and washed sequentially with water (200 mL) and
brine (300 mL). The organic layer was separated, dried (MgSO4),
filtered, and concentrated, and the resulting brown solid was purified
by flash column chromatography (elution: 5% EtOAc, 95% heptane)
to give 4-chloro-6-(3,4-dichlorophenyl)pyrimidine (3.6 g, 58% yield)
as a white solid. MS (ES+) m/z (M + 1) 261.
modulating KMO activity systemically contributes to disease
progression. Clearly, the lack of brain exposure of our lead
molecule does not allow us to test the hypothesis of whether
lowering 3HK and QA levels in situ in the brain is beneficial in
the context of HD. However, compound 75 does modulate the
pathway beautifully in the periphery in rodents and therefore
allows us to test whether a systemic, peripheral modulation of
the pathway affects disease progression, as has been claimed
previously.25 In addition, we clearly show that we can elevate
KYN, KYNA, and AA levels in brain. We believe that the
central elevation of KYN and KYNA is beneficial in the context
of the synaptic alterations observed in HD models. The dose-
dependency of the reponse in plasma reflects the existence of a
clear PK/PD response, which will be useful in clinical
development for this class of modecules, in the context of
HD or other indications where deregulation of KMO activity
has been implicated.
EXPERIMENTAL PROCEDURES
■
General Synthetic Procedures. Commercially available reagents
and solvents (HPLC grade) were used without further purification. 1H
and 13C NMR spectra were recorded on a Bruker DRX 500 MHz
spectrometer or Bruker DPX 250 MHz spectrometer in deuterated
solvents. Chemical shifts (δ) are in parts per million. Thin-layer
chromatography (TLC) analysis was performed with Kieselgel 60 F254
(Merck) plates and visualized using UV light.
Analytical HPLC-MS was performed on Shimadzu LCMS-2010EV
systems using reverse phase Atlantis dC18 columns (3 μm, 2.1 × 50
mm), gradient 5−100% B (A = water/0.1% formic acid, B =
acetonitrile/0.1% formic acid) over 3 min, injection volume 3 μL, flow
= 1.0 mL/min. UV spectra were recorded at 215 nm using a Waters
Pd(dppf)2Cl2 (0.46 g, 0.56 mmol) was added in one portion to a
stirred solution of 4-chloro-6-(3,4-dichlorophenyl)pyrimidine (2.9 g,
11.2 mmol) and triethylamine (3.1 mL, 22.4 mmol) in methanol (150
mL) within an autoclave. The autoclave was purged with nitrogen
(×2) before being sealed and charged with carbon monoxide (5 bar
P
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
pressure), and the reaction mixture was then heated to 50 °C and
stirred at this temperature and pressure for 18 h. After this time, the
reaction mixture was cooled to room temperature and concentrated.
The resulting residue was suspended in EtOAc (300 mL) and washed
sequentially with water (200 mL) and brine (300 mL). The organic
layer was separated, dried (MgSO4), filtered, and concentrated, and
the resulting brown solid was purified by flash column chromatog-
raphy (elution: 40% EtOAc, 60% heptane) to give methyl 6-(3,4-
dichlorophenyl)pyrimidine-4-carboxylate (2.2 g, 70% yield) as a white
solid. MS (ES+) m/z (M + 1) 284.
7.56 (m, 1H), 7.53−7.37 (m, 1H). 13C NMR (63 MHz, d4-AcOH)
167.42 (d, J = 2.70 Hz), 167.34, 164.98 (d, J = 245.53 Hz), 160.25,
156.45, 139.43 (d, J = 7.78 Hz), 132.59 (d, J = 8.14 Hz), 125.14 (d, J =
2.91 Hz), 120.38 (d, J = 21.52 Hz), 118.42, 116.04 (d, J = 23.62 Hz).
HRMS (ES+) m/z 219.0579 (219.057 Calcd for C11H7FN2O2 M + H).
6-(3-Chloro-4-fluorophenyl)pyrimidine-4-carboxylic Acid (55).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 253; H NMR (500 MHz,
DMSO) 9.31−9.36 (m, 1H), 8.53−8.57 (m, 1H), 8.40−8.46 (m, 1H),
8.21−8.27 (m, 1H), 7.44 (s, 1H). 13C NMR (126 MHz, DMSO)
163.4, 162.6, 159.28 (d, J = 252 Hz), 159.0, 157.1, 133.32 (d, J = 3
Hz), 129.7, 128.43 (d, J = 8 Hz, H), 120.71 (d, J = 18 Hz), 117.73 (d, J
= 21 Hz), 116.4. HRMS (ES+) m/z 253.0196 (253.0180 Calcd for
C11H6ClFN2O2 M + H).
NaOH (2 M solution, 5.0 mL, 10.0 mmol) was added in one
portion to a stirred solution of methyl 6-(3,4-dichlorophenyl)-
pyrimidine-4-carboxylate (0.95 g, 3.4 mmol) in THF (10 mL), and
the mixture was stirred at room temperature for 16 h. After this time,
the resulting precipitate was collected by filtration and washed with
water (1 mL) and DCM (20 mL) before being dried under vacuum to
give the sodium salt of 7 (0.64 g, 66% yield) as a white solid. MS (ES+)
6-(4-Chloro-3-fluorophenyl)pyrimidine-4-carboxylic Acid (56).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 253; H NMR (500 MHz,
1
m/z (M + 1) 269/271; H NMR (500 MHz, DMSO) 9.09−9.23 (m,
DMSO) 9.41 (d, J = 1.00 Hz, 1H), 8.60−8.52 (m, 1H), 8.31 (dd, J =
1.84, 10.65 Hz, 1H), 8.17 (dd, J = 1.54, 8.45 Hz, 1H), 7.78 (t, J = 8.06
Hz, 1H). 13C NMR (126 MHz, DMSO) 165.34, 162.64, 159.03,
157.73 (d, J = 247 Hz) 157.21, 136.62 (d, J = 7.0 Hz), 131.47, 124.53
(d, J = 3.4 Hz), 123.01 (d, J = 17.6 Hz), 116.53, 115.54 (d, J = 22.8
Hz). HRMS (ES+) m/z 253.0197 (253.018 Calcd for C11H6ClFN2O2
M + H).
1H), 8.18−8.23 (m, 1H), 8.13−8.17 (m, 1H), 7.85−7.92 (m, 1H),
7.60−7.70 (m, 1H). 13C NMR (126 MHz, CD3CO2D) 167.16, 165.74,
159.76, 156.68, 137.05, 136.82, 134.44, 132.27, 130.58, 128.12, 117.72.
HRMS (ES+) m/z 268.9877 (268.9885 Calcd for C11H6Cl2N2O2 M +
H).
6-(3-Chlorophenyl)pyrimidine-4-carboxylic Acid (6). This material
was synthesized according to general procedure A as described above.
MS (ES+) m/z (M + 1) 235/237; 1H NMR (500 MHz, D2O) 9.06 (s,
1H), 8.07 (s, 1H), 7.86 (s, 1H), 7.79 (d, J = 7.72 Hz, 1H), 7.37−7.47
(m, 2H). 13C NMR (126 MHz, D2O) 170.82, 164.73, 161.79, 157.80,
137.55, 134.49, 131.15, 130.56, 127.27, 125.78, 116.61. HRMS (ES+)
m/z 235.0255 (235.0274 Calcd for C11H7ClN2O2 M + H).
6-Phenylpyrimidine-4-carboxylic Acid (46). This material was
synthesized according to general procedure A as described above. MS
(ES+) m/z (M + 1) 201; 1H NMR (500 MHz, DMSO) 9.04−9.17 (m,
1H), 8.07−8.26 (m, 3H), 7.43−7.64 (m, 3H). 13C NMR (63 MHz,
CD3CO2D) 168.22, 167.41, 159.32, 156.46, 136.42, 133.05, 130.18,
128.78, 117.73. HRMS (ES+) m/z 201.0656 (201.0664 Calcd for
C11H8N2O2 M + H).
6-(3-Chloro-2-fluorophenyl)pyrimidine-4-carboxylic Acid (57).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 253; H NMR (500 MHz,
MeOD) 9.35−9.40 (m, 1H), 8.44−8.49 (m, 1H), 8.05−8.12 (m, 1H),
7.65−7.73 (m, 1H), 7.33−7.41 (m, 1H). HRMS (ES+) m/z 253.0176
(253.018 Calcd for C11H6ClFN2O2 M + H).
6-(3,4-Difluorophenyl)pyrimidine-4-carboxylic Acid (58). This
material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 236; H NMR (500 MHz,
DMSO) 9.10 (s, 1H), 8.22−8.28 (m, 1H), 8.19 (s, 1H), 8.06−8.11
(m, 1H), 7.54−7.65 (m, 1H). HRMS (ES+) m/z 237.0469 (237.0476
Calcd for C11H6F2N2O2 M + H).
6-(2,4-Difluorophenyl)pyrimidine-4-carboxylic Acid (59). This
6-(Pyridin-3-yl)pyrimidine-4-carboxylic Acid (47). This material
was synthesized according to general procedure A as described above.
MS (ES+) m/z (M + 1) 202; 1H NMR (500 MHz, DMSO) 9.33 (d, J
= 1.80 Hz, 1H), 9.14 (d, J = 1.25 Hz, 1H), 8.71 (dd, J = 1.54, 4.75 Hz,
1H), 8.53 (dt, J = 1.94, 7.99 Hz, 1H), 8.23 (d, J = 1.12 Hz, 1H), 7.56
(dd, J = 4.87, 7.91 Hz, 1H). HRMS (ES+) m/z 202.0607 (202.0617
Calcd for C10H7N3O2 M + H).
material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 237; H NMR (500 MHz,
MeOD) 9.48 (s, 1H), 8.51 (s, 1H), 8.34−8.26 (m, 1H), 7.23−7.16 (m,
2H). 13C NMR (126 MHz, DMSO) 165.2, 164.1 (dd, J = 13, 252 Hz),
161.3 (dd, J = 13, 255 Hz), 160.7 (d, J = 3 Hz), 159.1, 156.5, 132.4
(dd, J = 4, 10 Hz), 120.6 (dd, J = 3.53, 10.39 Hz), 119.7 (d, J = 11
Hz), 112.8 (dd, J = 3, 22 Hz), 105.3 (t, J = 27 Hz). HRMS (ES+) m/z
237.0472 (237.0476 Calcd for C11H6F2N2O2 M + H).
6-(3-Phenylphenyl)pyrimidine-4-carboxylic Acid (48). This ma-
terial was synthesized according to general procedure A as described
6-(4-Chloro-2-fluorophenyl)pyrimidine-4-carboxylic Acid (60).
1
above. MS (ES+) m/z (M + 1) 277; H NMR (500 MHz, DMSO)
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 253/255; H NMR (500
13.79 (br s, 1H), 9.43 (d, J = 1.10 Hz, 1H), 8.63 (d, J = 1.10 Hz, 1H),
8.52 (s, 1H), 8.28 (d, J = 7.88 Hz, 1H), 7.90 (d, J = 7.88 Hz, 1H),
7.76−7.86 (m, 2H), 7.68 (t, J = 7.80 Hz, 1H), 7.52 (t, J = 7.65 Hz,
2H), 7.37−7.46 (m, 1H). HRMS (ES+) m/z 277.0979 (277.0977
Calcd for C17H12N2O2 M + H).
MHz, MeOD) 9.38 (br. s, 1H), 8.26 (br. s, 1H.), 8.18 (t, J = 8.54 Hz,
1H), 7.70 (dd, J = 11.44, 1.83 Hz, 1H), 7.52 (dd, J = 8.47, 1.75 Hz,
1H). HRMS (ES+) m/z 253.0179 (253.018 Calcd for C11H6ClFN2O2
M + H).
6-(4-Chlorophenyl)pyrimidine-4-carboxylic Acid (52). This ma-
terial was synthesized according to general procedure A as described
above. MS (ES+) m/z (M + 1) 235/237; 1H NMR (500 MHz,
DMSO) 9.34−9.46 (m, 1H), 8.45−8.56 (m, 1H), 8.25−8.38 (m, 2H),
7.57−7.71 (m, 2H). 13C NMR (126 MHz, DMSO) 165.43, 163.71,
159.06, 156.89, 136.65, 134.31, 129.28, 129.18, 116.19. HRMS (ES+)
m/z 235.0269 (235.0274 Calcd for C11H7ClN2O2 M + H).
6-[3-Chloro-4-(trifluoromethyl)phenyl]pyrimidine-4-carboxylic
Acid (61). This material was synthesized according to general
1
procedure A as described above. MS (ES+) m/z (M + 1) 303; H
NMR (250 MHz, DMSO) 9.43−9.52 (m, 1H), 8.63−8.69 (m, 1H),
8.58 (s, 1H), 8.39−8.47 (m, 1H), 7.99−8.08 (m, 1H). 13C NMR (126
MHz, DMSO) 165.3, 162.1, 159.1, 157.4, 141.0, 131.8, 130.1, 128.8−
128.7 (m), 128.6, 126.6, 122.7 (q, J = 273 Hz). 117.3. HRMS (ES+)
m/z 303.0129 (303.0148 Calcd for C12H6ClF3N2O2 M + H).
6-[3-(Trifluoromethyl)phenyl]pyrimidine-4-carboxylic Acid (62).
This material was synthesized according to general procedure A as
6-(3,5-Dichlorophenyl)pyrimidine-4-carboxylic Acid (53). This
material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 269/271; H NMR (500
1
described above. MS (ES+) m/z (M + 1) 269; H NMR (500 MHz,
MHz, DMSO) 9.44 (s, 1H), 8.63 (s, 1H), 8.33 (s, 2H), 7.86 (s, 1H).
13C NMR (126 MHz, DMSO) 165.29, 162.17, 159.00, 157.37, 138.99,
135.11, 130.86, 126.03, 117.01. HRMS (ES+) m/z 268.9887 (268.9885
Calcd for C11H6Cl2N2O2 M + H).
DMSO) 9.45 (s, 1H), 8.57−8.68 (m, 3H), 7.94−8.00 (m, 1H), 7.80−
7.86 (m, 1H). HRMS (ES+) m/z 269.0529 (269.0538 Calcd for
C12H7F3N2O2 M + H).
6-(3-Fluorophenyl)pyrimidine-4-carboxylic Acid (54). This ma-
6-[2-(Trifluoromethyl)phenyl]pyrimidine-4-carboxylic Acid (63).
terial was synthesized according to general procedure A as described
This material was synthesized according to general procedure A as
above. MS (ES+) m/z (M + 1) 219; H NMR (500 MHz, DMSO)
described above. MS (ES+) m/z (M + 1) 269; H NMR (500 MHz,
1
1
14.01 (br s, 1H), 9.42 (s, 1H), 8.54 (s, 1H), 8.23−8.00 (m, 2H), 7.71−
CDCl3) 9.47 (s, 1H), 8.28 (s, 1H), 7.89−7.82 (m, 1H), 7.72−7.66 (m,
Q
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 7.56−7.53 (1H, m). 13C NMR (126 MHz, DMSO) 166.76,
165.23, 158.39, 156.60, 136.57−136.55 (m), 132.80, 131.58, 130.34,
126.89−126.72 (m), 126.77 (q, J = 30.88 Hz), 123.90 (q, J = 273.73
Hz), 120.13. HRMS (ES+) m/z 269.0537 (269.0538 Calcd for
C12H7F3N2O2 M + H).
(126 MHz, DMSO) 168.22, 165.56, 162.52, 157.64, 136.28, 134.22,
132.16, 131.34, 129.04, 127.42, 113.52, 25.80. HRMS (ES+) m/z
283.0038 (283.0041 Calcd for C12H8Cl2N2O2 M + H).
6-(3,4-Dichlorophenyl)-5-fluoropyrimidine-4-carboxylic Acid
(23). This material was synthesized according to general procedure
1
6-(3-Methylphenyl)pyrimidine-4-carboxylic Acid (64). This ma-
A as described above. MS (ES+) m/z (M + 1) 287; H NMR (500
terial was synthesized according to general procedure A as described
MHz, DMSO) 9.20 (d, J = 2.29 Hz, 1H), 8.24 (d, J = 1.83 Hz, 1H),
7.98−8.05 (m, 1H), 7.88 (d, J = 8.39 Hz, 1H). HRMS (ES+) m/z
286.9794 (286.979 Calcd for C11H5Cl2FN2O2 M + H).
1
above. MS (ES+) m/z (M + 1) 215; H NMR (500 MHz, DMSO)
13.88 (br. s, 1H), 9.37 (d, J = 1.06 Hz, 1H), 8.46 (d, J = 1.05 Hz, 1H),
8.16−7.83 (m, 2H), 7.51−7.19 (m, 2H), 2.41 (s, 3H). 13C NMR (126
MHz, DMSO) 165.52, 164.97, 159.00, 156.70, 138.60, 135.40, 132.40,
129.14, 127.79, 124.45, 116.10, 21.00. HRMS (ES+) m/z 215.0806
(215.0821 Calcd for C12H10N2O2 M + H).
6-(3,4-Dichlorophenyl)-5-methoxypyrimidine-4-carboxylic Acid
(27). This material was synthesized according to general procedure
A as described above. MS (ES+) m/z (M + 1) 299/301; 1H NMR (500
MHz, DMSO) 8.73 (s, 1H) 8.21 (d, J = 1.89 Hz, 1H) 8.02 (dd, J =
8.51, 2.05 Hz, 1H) 7.78 (d, J = 8.51 Hz, 1H) 3.64−3.83 (m, 3H). 13C
NMR (63 MHz, DMSO) 167.65, 163.65, 152.59, 152.46, 146.33,
136.26, 132.45, 131.20, 130.74, 130.55, 129.13, 60.72. HRMS (ES+)
m/z 298.9988 (298.999 Calcd for C12H8Cl2N2O3 M + H).
6-(3,4-Dichlorophenyl)-5-methylpyrimidine-4-carboxylic Acid
(25). This material was synthesized according to general procedure
A as described above. MS (ES+) m/z (M + 1) 283/285; 1H NMR (500
MHz, DMSO) 14.13 (br s, 1H) 9.16 (s, 1H) 7.94 (d, J = 2.05 Hz, 1H)
7.81 (d, J = 8.35 Hz, 1H) 7.67 (dd, J = 8.35, 2.05 Hz, 1H) 2.37 (s,
3H). 13C NMR (126 MHz, DMSO) 166.98, 164.37, 158.94, 155.55,
137.93, 132.52, 131.31, 131.08, 130.64, 129.45, 125.40, 15.22. HRMS
(ES+) m/z 283.0052 (283.0041 Calcd for C12H8Cl2N2O2 M + H).
5-Amino-6-(3,4-dichlorophenyl)pyrimidine-4-carboxylic Acid
(24). This material was synthesized according to general procedure
A as described above. MS (ES+) m/z (M + 1) 284/286; 1H NMR (500
MHz, DMSO) 8.36 (s, 1H) 7.92 (d, J = 1.58 Hz, 1H) 7.68−7.78 (m,
2H) 6.87 (br s, 2H). HRMS (ES+) m/z 283.9976 (283.9994 Calcd for
C11H7Cl2N3O2 M + H).
General Method B. 6-(3-Chloro-4-cyclopropoxyphenyl)-
pyrimidine-4-carboxylic Acid (75). Bromocyclopropane (87.5 g,
0.723 mol) was added portionwise over 10 min to a stirred solution
of 4-bromo-2-chlorophenol (30.0 g, 0.145 mol) and cesium
dicarbonate (118 g, 0.362 mol) in dimethylacetamide (450 mL).
The mixture was heated to 150 °C and stirred at this temperature for
16 h. After this time, the reaction was cooled to room temperature,
poured onto ice−water (600 mL), and extracted with TBME (3 × 400
mL). The organic layers were combined and washed with water (2 ×
200 mL) before being dried (MgSO4), filtered, and concentrated to
give 4-bromo-2-chloro-1-cyclopropoxybenzene (31.2 g, 72% yield) as a
pale orange oil which was used directly without purification. 1H NMR
(500 MHz, CDCl3) 7.51−7.45 (m, 1H), 7.34 (dd, J = 8.8, 2.4 Hz, 1H),
7.21−7.13 (m, 1H), 3.84−3.74 (m, 1H) 0.93−0.77 (m, 4H).
6-[4-Fluoro-3-(trifluoromethyl)phenyl]pyrimidine-4-carboxylic
Acid (65). This material was synthesized according to general
1
procedure A as described above. MS (ES+) m/z (M + 1) 287; H
NMR (500 MHz, DMSO) 14.04 (br. s, 1H), 9.42 (s, 1H), 8.79−8.42
(m, 3H), 7.71 (t, J = 9.61 Hz, 1H). 13C NMR (126 MHz, DMSO)
165.4, 162.4, 160.79 (d, J = 258.91 Hz, H), 159.0, 157.2, 134.48 (d, J =
9.62 Hz, H), 132.6, 126.4, 122.43 (q, J = 271.93 Hz, H), 118.27 (d, J =
20.96 Hz, H), 117.45 (dd, J = 12.80, 32.66 Hz, H), 116.6. HRMS
(ES+) m/z 287.044 (287.0444 Calcd for C12H6F4N2O2 M + H).
6-(3-Chloro-4-methylphenyl)pyrimidine-4-carboxylic Acid (66).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 249; H NMR (500 MHz,
DMSO) 9.39 (d, J = 1.37 Hz, 1H), 8.52 (d, J = 1.22 Hz, 1H), 8.32 (d, J
= 1.83 Hz, 1H), 8.17 (dd, J = 1.83, 7.93 Hz, 1H), 7.56 (d, J = 8.09 Hz,
1H), 2.42 (s, 3H). HRMS (ES+) m/z 249.0442 (249.0431 Calcd for
C12H9ClN2O2 M + H).
6-(3-Fluoro-4-methylphenyl)pyrimidine-4-carboxylic Acid (67).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 232; H NMR (500 MHz,
DMSO) 9.38 (s, 1H) 8.49 (s, 1H) 7.94−8.11 (m, 2H) 7.39−7.57 (m,
1H) 2.32 (s, 3H). 13C NMR (126 MHz, DMSO) 165.46, 163.59,
161.14 (d, J = 243.39 Hz), 158.99, 156.89, 135.33 (d, J = 7.76 Hz),
132.44 (d, J = 5.18 Hz), 128.45 (d, J = 17.24 Hz), 123.15 (d, J = 3.01
Hz), 116.09, 113.49 (d, J = 24.08 Hz), 14.31 (d, J = 2.98 Hz). HRMS
(ES+) m/z 233.0744 (233.0726 Calcd for C12H9FN2O2 M + H).
6-(4-Chloro-3-methoxyphenyl)pyrimidine-4-carboxylic Acid (68).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 265/267; H NMR (500
MHz, DMSO) 14.01 (br s, 1H), 9.41 (d, J = 1.26 Hz, 1H), 8.58 (d, J =
1.10 Hz, 1H), 7.97 (d, J = 1.89 Hz, 1H), 7.90 (dd, J = 2.05, 8.35 Hz,
1H), 7.63 (d, J = 8.35 Hz, 1H), 4.00 (s, 3H). 13C NMR (126 MHz,
DMSO) 165.51, 163.83, 158.95, 156.93, 155.05, 135.71, 130.55,
124.77, 120.45, 116.36, 111.01, 56.38. HRMS (ES+) m/z 265.0381
(265.038 Calcd for C12H9ClN2O3 M + H).
Potassium acetate (37.1 g, 0.378 mol) was added portionwise over
10 min to a stirred solution of 4-bromo-2-chloro-1-cyclopropox-
ybenzene (31.2 g, 0.126 mol) and bis(pinacolato)diboron (38.4 g,
0.151 mol) in DMSO (340 mL). The mixture was degassed with
nitrogen for 15 min, after which time Pd(dppf)2Cl2 (4.60 g, 6.28
mmol) was added in one portion, and the mixture was then heated to
80 °C and stirred at this temperature for 16 h under a nitrogen
atmosphere. After this time, the reaction mixture was cooled to room
temperature and partitioned between ethyl acetate (300 mL) and
water (300 mL). The organic layer was removed and washed
sequentially with water (1.50 L) and then brine (500 mL) before being
dried (MgSO4), filtered, and concentrated. The resulting residue was
dry-loaded onto silica gel (90.0 g) and purified by dry flash column
chromatography (elution: 100% heptane to 92% heptane, 8% EtOAc)
to give 2-(3-chloro-4-cyclopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-
6-(3-Chloro-4-methoxyphenyl)pyrimidine-4-carboxylic Acid (73).
This material was synthesized according to general procedure A as
1
described above. MS (ES+) m/z (M + 1) 265; H NMR (500 MHz,
DMSO) 13.92 (br s, 1H), 9.27−9.36 (m, 1H), 8.47 (d, J = 1.22 Hz,
1H), 8.35 (d, J = 2.14 Hz, 1H), 8.27 (dd, J = 2.29, 8.70 Hz, 1H), 7.21−
7.36 (m, 1H), 3.96 (s, 3H). 13C NMR (126 MHz, DMSO) 165.54,
163.26, 158.92, 157.26, 156.67, 128.63 (2 peaks), 127.85, 122.01,
115.60, 113.16, 56.55. HRMS (ES+) m/z 265.0389 (265.038 Calcd for
C12H9ClN2O3 M + H).
6-[3-Chloro-4-(propan-2-yloxy)phenyl]pyrimidine-4-carboxylic
Acid (74). This material was synthesized according to general
procedure A as described above. MS (ES+) m/z (M + 1) 293/295;
1H NMR (500 MHz, DMSO) 9.33 (s, 1H), 8.47 (s, 1H), 8.36 (d, J =
1.94 Hz, 1H), 8.24 (dd, J = 1.89, 8.72 Hz, 1H), 7.34 (d, J = 8.80 Hz,
1H), 4.84 (p, J = 5.98 Hz, 1H), 1.34 (d, J = 6.00 Hz, 5H). 13C NMR
(63 MHz, DMSO) 166.43, 165.79, 161.58, 158.21, 155.15, 129.43,
128.46, 127.12, 122.90, 115.22, 114.17, 71.36, 21.81. HRMS (ES+) m/
z 293.0679 (293.0693 Calcd for C14H13ClN2O3 M + H).
1
dioxaborolane (27.0 g, 62% yield) as a pale yellow oil. H NMR (500
MHz, CDCl3) 7.79 (d, J = 1.3 Hz, 1 H) 7.67 (dd, J = 8.2, 1.4 Hz, 1 H)
7.32−7.25 (m, 1 H) 3.90−3.77 (m, 1 H) 1.42−1.30 (m, 12 H) 0.95−
0.77 (m, 4 H).
2-(3-Chloro-4-cyclopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane was then taken forward as described in general procedure A
6-(3,4-Dichlorophenyl)-2-methylpyrimidine-4-carboxylic Acid
(22). This material was synthesized according to general procedure
1
above to give compound 75. MS (ES+) m/z (M + 1) 291/293; H
1
A as described above. MS (ES+) m/z (M + 1) 283; H NMR (500
NMR (500 MHz, DMSO) 9.10−9.05 (m, 1H), 8.28−8.17 (m, 3H),
MHz, DMSO) 8.49 (d, J = 1.98 Hz, 1H), 8.37 (s, 1H), 8.25 (dd, J =
8.53, 2.13 Hz, 1H), 7.81 (d, J = 8.38 Hz, 1H), 2.76 (s, 3H). 13C NMR
7.60−7.54 (m, 1H), 4.11−4.02 (m, 1H) 0.92−0.71 (m, 4H). 13C
R
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
NMR (300 MHz, DMSO) 166.7, 166.4, 161.8, 158.5, 156.6, 130.5,
128.6, 127.5, 122.1, 115.3, 114.5, 52.5, 6.4.
g, 13.9 mmol) in 20 mL of 15% HCl at −5 °C. The solid material was
removed by filtration, and a solution of sodium tetrafluoroborate (2.45
g, 22.3 mmol) in water (5 mL) was mixed with the filtrate. The
resulting solid was collected by filtration, washed with water (5 mL),
and dried on a sinter funnel under vacuum for 1 h. It was then dried in
the vacuum oven at 40 °C until constant weight to give 3-chloro-4-
(trifluoromethoxy)benzene-1-diazonium tetrafluoroboranide (2.93 g,
68% yield) as a white solid. MS (ES+) m/z (M + 1) 223/225.
Bis(pinacolato) diboron (2.51 g, 9.8 mmol) was added in one
portion to a cooled (0 °C), stirred suspension of 3-chloro-4-
(trifluoromethoxy)benzene-1-diazonium tetrafluoroboranide (2.92 g,
9.41 mmol) in methanol (20 mL). The mixture was degassed with
nitrogen for 10 min before PdCl2(dppf)2 (0.19 g, 0.23 mmol) was
added, and the mixture was stirred at room temperature for 18 h. After
this time, the reaction was evaporated to dryness, redissolved in DCM,
dry loaded onto silica, and purified by dry flash chromatography
(elution: 100% heptane to 20% EtOAc, 80% heptane) to give 2-[3-
chloro-4-(trifluoromethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (1.11 g, 37% yield) as a brown oil which was taken on
directly as described in general procedure A above to give compound
6-(3-Chloro-4-cyclobutoxyphenyl)pyrimidine-4-carboxylic Acid
(78). This material was synthesized according to general procedure
B as described above. MS (ES+) m/z (M + 1) 305/307; 1H NMR (500
MHz, MeOD) 9.20−9.29 (m, 1H) 8.40−8.49 (m, 1H) 8.25−8.34 (m,
1H) 8.07−8.18 (m, 1H) 7.04−7.11 (m, 1H) 2.48−2.62 (m, 2H)
2.16−2.28 (m, 2H) 1.87−1.96 (m, 1H) 1.73−1.83 (m, 1H). 13C NMR
(126 MHz, DMSO) 165.52, 163.27, 158.90, 156.64, 155.22, 128.83,
128.61, 127.75, 122.19, 115.56, 114.38, 72.07, 29.97, 12.72. HRMS
(ES+) m/z 305.0697 (305.0693 Calcd for C15H13ClN2O3 M + H).
6-[3-Chloro-4-(oxetan-3-yloxy)phenyl]pyrimidine-4-carboxylic
Acid (79). This material was synthesized according to general
procedure B as described above. MS (ES+) m/z (M + 1) 307/309;
δH (500 MHz, DMSO) 9.12 (s, 1H), 8.32 (d, J = 2.05 Hz, 1H), 8.27
(s, 1H), 8.13 (dd, J = 2.05, 8.67 Hz, 1H), 6.94 (d, J = 8.67 Hz, 1H),
5.48 (quin, J = 5.40 Hz, 1H), 5.00 (t, J = 6.78 Hz, 2H), 4.62 (dd, J =
4.89, 7.41 Hz, 2H). 13C NMR (63 MHz, d4-AcOH) 167.72, 167.00,
159.85, 157.02, 156.69, 131.60, 131.13, 129.54, 125.33, 117.64, 114.94,
79.21, 72.88. HRMS (ES+) m/z 307.0492 (307.0486 Calcd for
C14H11ClN2O4 M + H).
1
72. MS (ES+) m/z (M + 1) 319/321; H NMR (500 MHz, DMSO)
6-[3-Chloro-4-(cyclopentyloxy)phenyl]pyrimidine-4-carboxylic
9.45 (s, 1H), 8.58−8.65 (m, 2H), 8.37−8.44 (m, 1H), 7.74−7.81 (m,
1H). HRMS (ES+) m/z 319.0091 (319.0097 Calcd for
C12H6ClF3N2O3 M + H).
Acid (80). This material was synthesized according to general
1
procedure B as described above. MS (ES+) m/z (M + 1) 319; H
NMR (500 MHz, DMSO) 9.33 (s, 1H), 8.47 (s, 1H), 8.37−8.29 (m,
1H), 8.29−8.17 (m, 1H), 7.31 (d, J = 8.77 Hz, 1H), 5.05 (s, 1H),
2.10−1.87 (m, 2H), 1.84−1.53 (m, 7H). 13C NMR (126 MHz,
DMSO) 165.58, 163.26, 158.90, 156.81, 155.81, 128.79, 128.30,
127.58, 122.90, 115.50, 115.05, 80.60, 32.29, 23.61. HRMS (ES+) m/z
319.0852 (319.0849 Calcd for C16H15ClN2O3 M + H).
6-[3-Chloro-4-(cyclohexyloxy)phenyl]pyrimidine-4-carboxylic
Acid (81). This material was synthesized according to general
procedure B as described above. MS (ES+) m/z (M + 1) 333/335;
1H NMR (500 MHz, DMSO) 9.04 (s, 1H), 8.23 (d, J = 2.18 Hz, 1H),
8.15−8.07 (m, 2H), 7.33 (d, J = 8.84 Hz, 1H), 4.60 (dt, J = 4.35, 8.15
Hz, 1H), 1.96−1.88 (m, 2H), 1.79−1.69 (m, 2H), 1.61−1.48 (m, 3H),
1.47−1.28 (m, 3H). 13C NMR (63 MHz, d4-AcOH) 168.14, 167.36,
159.54, 158.40, 157.09, 131.43, 129.77, 129.40, 126.12, 117.51, 116.39,
78.07, 32.74, 26.93, 24.60. HRMS (ES+) m/z 333.1006 (333.1006
Calcd for C17H17ClN2O3 M + H).
6-[3-Chloro-4-(pyrrolidin-1-yl)phenyl]pyrimidine-4-carboxylic
Acid (71). 4-Bromo-2-chloroaniline (2.0 g, 9.69 mmol), 1,4-
dibromobutane (2.31 mL, 19.4 mmol), potassium carbonate (2.68 g,
19.4 mmol), water (25 mL), and dioxane (10 mL) were heated to 100
°C overnight with vigorous stirring. The reaction mixture was allowed
to cool and then extracted with EtOAc (2 × 25 mL). The combined
organics were washed with brine (15 mL), dried (MgSO4), filtered,
and concentrated. The resulting residue was purified by flash column
chromatography (elution: 100% heptane to 20% EtOAc, 80%
heptane) to give 1-(4-bromo-2-chlorophenyl)pyrrolidine (1.16 g,
1
45% yield) as a yellow oil. MS (ES+) m/z (M + 1) 260/262. H
NMR (500 MHz, DMSO) 7.48 (d, J = 2.36 Hz, 1H), 7.33 (dd, J =
2.36, 8.83 Hz, 1H), 6.87 (d, J = 8.83 Hz, 1H), 3.29−3.33 (m, 4H),
1.87 (td, J = 3.43, 6.38 Hz, 4H). This material was then taken on as
described in general procedure A above to give the title compound 71.
MS (ES+) m/z (M + 1) 304/306; 1H NMR (500 MHz, DMSO) 9.25
(s, 1H), 8.37 (s, 1H), 8.24 (s, 1H), 8.10 (s,1H), 6.98 (d, J = 8.9 Hz,
1H), 3.52 (t, J = 6.4 Hz, 5H), 1.92 (t, J = 6.5 Hz, 4H). 13C NMR (63
MHz, DMSO) 165.85, 163.20, 158.78, 157.19, 148.60, 130.12, 126.78,
125.32, 119.75, 116.57, 114.52, 50.82, 25.27. HRMS (ES+) m/z
304.0855 (304.0853 Calcd for C15H14ClN3O2 M + H).
6-{3-Chloro-4-[2-(morpholin-4-yl)ethoxy]phenyl}pyrimidine-4-
carboxylic Acid (69). This material was synthesized according to
general procedure B as described above. MS (ES+) m/z (M + 1) 364/
1
366; H NMR (500 MHz, D2O) 8.90−9.04 (m, 1H), 7.93−8.02 (m,
1H), 7.80−7.89 (m, 1H), 7.69−7.78 (m, 1H), 6.92−7.06 (m, 1H),
4.10−4.25 (m, 2H), 3.55−3.78 (m, 4H), 2.78−2.90 (m, 2H), 2.55−
2.74 (m, 4H). HRMS (ES+) m/z 364.1081 (364.1064 Calcd for
C17H18ClN3O4 M + H).
6-(3,4-Dichlorophenyl)-5-(methoxymethyl)pyrimidine-4-carbox-
ylic Acid (26). Methyl 6-(3,4-dichlorophenyl)-5-methylpyrimidine-4-
carboxylate, N-bromosuccinimide (0.66 g, 4.0 mmol), and AIBN (0.05
g, 0.34 mmol) were dissolved in 1,2-dichloroethane, heated to 90 °C
under a nitrogen atmosphere, and stirred at this temperature for 18 h.
After this time, the reaction was cooled to room temperature, saturated
aqueous sodium bicarbonate solution (20 mL) was added, and the
organic layer was separated. The organic layer was diluted with DCM
(20 mL) and then washed with a further 20 mL of saturated aqueous
sodium bicarbonate solution, followed by water (100 mL) and brine
(50 mL). The organic layer was separated, dried (MgSO4), filtered,
and concentrated. The resulting residue was purified by flash column
chromatography (elution: 5% methanol, 95% DCM) to give methyl 5-
(bromomethyl)-6-(3,4-dichlorophenyl)pyrimidine-4-carboxylate (0.46
6-{4-Chloro-3-[2-(morpholin-4-yl)ethoxy]phenyl}pyrimidine-4-
carboxylic Acid (70). This material was synthesized according to
general procedure B as described above. MS (ES+) m/z (M + 1) 364/
1
366; H NMR (500 MHz, DMSO) 9.41 (d, J = 0.96 Hz, 1H), 8.63−
8.40 (m, 1H), 8.11−7.83 (m, 2H), 7.66 (d, J = 8.32 Hz, 1H), 4.89−
4.47 (m, 2H), 3.91 (s, 4H), 3.65−3.53 (m, 2H), 3.39 (s, 4H). 13C
NMR (126 MHz, DMSO) 165.49, 163.63, 158.98, 157.07, 153.54,
135.78, 130.75, 125.03, 121.27, 116.42, 112.15, 64.18, 63.39, 54.84,
51.99. HRMS (ES+) m/z 364.1063 (364.1064 Calcd for
C17H18ClN3O4 M + H).
6-[3-Chloro-4-(cyclopropylmethoxy)phenyl]pyrimidine-4-carbox-
ylic Acid (76). This material was synthesized according to general
procedure B as described above. MS (ES+) m/z (M + 1) 303/305. 1H
NMR (500 MHz, DMSO) 9.04 (s, 1 H) 8.25 (d, J=2.21 Hz, 1 H)
8.06−8.16 (m, 2 H) 7.27 (d, J = 8.67 Hz, 1 H) 4.02 (d, J = 7.09 Hz, 2
H) 1.24−1.37 (m, 1 H) 0.34−0.67 (m, 4 H). 13C NMR (63 MHz, d4-
AcOH) 167.86, 167.36, 159.56, 159.50, 156.69, 131.18, 129.86, 129.52,
125.20, 117.49, 115.00, 75.26, 11.34, 4.22. HRMS (ES+) m/z 305.0701
(305.0693 Calcd for C15H13ClN2O3 M + H).
General Method C. 6-[3-Chloro-4-(trifluoromethoxy)phenyl]-
pyrimidine-4-carboxylic Acid (72). A solution of sodium nitrite
(2.31 g, 33.4 mmol) in water (15 mL) was slowly added over 30 min
to a suspension of [3-chloro-4-(trifluoromethoxy)phenyl]amine (2.95
1
g, 36% yield) as a yellow oil which solidified on standing. H NMR
(500 MHz, DMSO) 9.33 (s, 1H) 7.95−8.01 (m, 1H) 7.85−7.91 (m,
1H) 7.69 (dd, J = 8.20, 2.05 Hz, 1H) 4.72−4.80 (m, 1H) 3.97−4.01
(m, 3H).
Sodium methoxide (0.03g, 0.53 mmol) was added in one portion to
a stirred solution of methyl 5-(bromomethyl)-6-(3,4-dichlorophenyl)-
pyrimidine-4-carboxylate (0.1 g, 0.26 mmol) in methanol (5 mL), and
the mixture was stirred at room temperature under a nitrogen
atmosphere for 20 h. After this time, the mixture was concentrated,
and the resulting residue taken up in DCM (10 mL). The solution was
washed consecutively with water (2 × 50 mL) and brine (2 × 50 mL),
S
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
before being separated, dried (MgSO4), filtered, and concentrated.
The resulting residue was purified by flash column chromatography
(elution: 100% DCM to 99% DCM: 1% methanol) to give methyl 6-
(3,4-dichlorophenyl)-5-(methoxymethyl)pyrimidine-4-carboxylate
(0.02g, 20% yield) as a white solid. MS (ES+) m/z (M + 1) 327/329.
Sodium hydroxide (0.05 mL, 0.1 mmol) was added in one portion
to a stirred solution of methyl 6-(3,4-dichlorophenyl)-5-
(methoxymethyl)pyrimidine-4-carboxylate (0.1 g, 0.26 mmol) in
THF (5 mL), and the mixture was stirred at room temperature
under a nitrogen atmosphere for 20 h. After this time, the resulting
precipitate was collected by filtration, washed with water (1 mL), and
dried under vacuum to give the title compound 26 (0.004 g, 15%
dichlorophenoxy)pyrimidine (0.32 g, 37% yield) as a white solid. MS
(ES+) m/z (M + 1) 274/276. This material was then taken on as
described in general procedure A above to give the title compound 13.
MS (ES+) m/z (M + 1) 285; 1H NMR (500 MHz, DMSO) 14.04 (br
s, 1H), 8.91 (s, 1H), 7.70−7.81 (m, 2H), 7.61 (s, 1H), 7.31−7.39 (m,
1H). 13C NMR (126 MHz, DMSO) 169.81, 164.75, 158.56, 158.09,
151.10, 131.84, 131.50, 128.52, 124.39, 122.63, 108.75. HRMS (ES+)
m/z 284.9821 (284.9834 Calcd for C11H6Cl2N2O3 M + H).
6-Cyclohexylpyrimidine-4-carboxylic Acid (15). Cyclohexylzinc
bromide (0.66 g, 2.9 mmol) was added in one portion to a stirred
solution of methyl 6-chloropyrimidine-4-carboxylate (0.5 g, 2.9 mmol)
and PdCl2(dppf)2 (0.12 g, 0.14 mmol) in THF (5 mL). The mixture
was degassed with nitrogen for 15 min before being heated to reflux
and stirred for 15 h. After this time, the reaction mixture was cooled to
room temperature and treated with aqueous saturated ammonium
chloride (25 mL), before being extracted with ethyl acetate (3 × 25
mL). The combined organic extracts were dried (Na2SO4), filtered,
and concentrated. The resulting residue was purified on a Biotage
machine (100 g column), eluting with ethyl acetate−heptane (7:93−
3CV, 7:93 to 60:40−10 CV, 40:60−2CV) to give methyl 6-
cyclohexylpyrimidine-4-carboxylate (0.13 g, 21% yield) as a pale
yellow oil. MS (ES+) m/z (M + 1) 221.
Sodium hydroxide solution (1.5 mL, 3.1 mmol, 2 M) was added in
one portion to a stirred solution of methyl 6-cyclohexylpyrimidine-4-
carboxylate (0.13 g, 0.61 mmol) in THF (3 mL), and the mixture was
stirred at room temperature under a nitrogen atmosphere for 2 h. After
this time, the resulting precipitate was collected by filtration, washed
with water (1 mL), and dried under vacuum to give the title
compound 15 (0.045 g, 35% yield) as a white solid. MS (ES+) m/z (M
+ 1) 207; 1H NMR (500 MHz, DMSO) 13.86 (br s, 1H) 9.23 (s, 1H)
7.87 (s, 1H) 2.70−2.88 (m, 1H) 1.12−1.97 (m, 10H). 13C NMR (126
MHz, DMSO) 176.30, 165.55, 158.47, 155.62, 118.26, 44.98, 31.37,
25.65, 25.40. HRMS (ES+) m/z 207.1123 (207.1134 Calcd for
C11H14N2O2 M + H).
6-(3,4-Dichlorophenyl)pyrimidine-4-carbonitrile (30). A mixture
of 4-chloro-6-(3,4-dichlorophenyl)pyrimidine (4.0 g, 15.4 mmol), zinc
cyanide (1.8 g, 15.3 mmol), Pd(PPh3)4 (0.89 g, 0.77 mmol), and DMF
(80 mL) was stirred at 100 °C under nitrogen for 19 h. After this time,
the reaction mixture was cooled and then shaken with brine (100 mL),
water (20 mL), and ethyl acetate (100 mL). The mixture was filtered
and the separated organic layer dried (MgSO4), filtered, and
concentrated. The resulting residue was purified by flash column
chromatography (elution: 6% EtOAc, 94% heptane) to give the title
compound (1.73 g, 45% yield) as a white solid. MS (ES+) m/z (M +
1) 250/252; 1H NMR (500 MHz, DMSO) 9.45 (d, J = 1.19 Hz, 1H),
8.89 (d, J = 1.22 Hz, 1H), 8.50 (d, J = 2.08 Hz, 1H), 8.25 (dd, J = 2.11,
8.49 Hz, 1H), 7.86 (d, J = 8.48 Hz, 1H). 13C NMR (126 MHz,
DMSO) 162.52, 159.46, 141.48, 135.21, 134.99, 132.36, 131.55,
129.28, 127.45, 121.43, 116.03. HRMS (ES+) m/z 249.9929 (249.9939
Calcd for C11H5Cl2N3 M + H).
1
yield) as a white solid. MS (ES+) m/z (M + 1) 313/315; H NMR
(500 MHz, DMSO) 8.95 (s, 1H) 7.99 (d, J = 1.73 Hz, 1H) 7.76−7.83
(m, 1H) 7.70−7.76 (m, 1H) 4.39 (s, 2H) 3.22 (s, 3H).
6-(3,4-Dichlorophenyl)-5-hydroxypyrimidine-4-carboxylic Acid
(28). Lithium chloride (0.19 g, 5.0 mmol) was added in one portion
to a stirred solution of methyl 6-(3,4-dichlorophenyl)-5-hydroxypyr-
imidine-4-carboxylate (0.47 g, 2.0 mmol) in DMF (5 mL) and the
mixture was heated to 145 °C and stirred at this temperature under a
nitrogen atmosphere for 18 h. After this time, the mixture was cooled
to room temperature and partitioned between ethyl acetate (50 mL)
and 1 M HCl solution (25 mL). The organic layer was separated and
washed consecutively with 1 M HCl solution (50 mL), water (50 mL),
and brine (50 mL) before being dried (MgSO4), filtered, and
concentrated. The resulting residue was purified by prep HPLC to give
the title compound 28 (0.02 g, 4% yield) as a yellow solid. MS (ES+)
1
m/z (M + 1) 285/287; H NMR (500 MHz, DMSO) 8.66−8.75 (m,
1H) 8.48−8.56 (m, 1H) 8.40−8.46 (m, 1H) 7.70−7.80 (m, 1H).
6-(Morpholin-4-yl)pyrimidine-4-carboxylic Acid (12). Morpholine
(1.23 mL, 14.1 mmol) was added in one portion to a suspension of
4,6-dichloropyrimidine in isopropanol (10 mL) in a pressure tube. The
tube was sealed, heated to 80 °C, and stirred at this temperature for 2
h. After this time, the tube was cooled to room temperature and the
reaction mixture was concentrated. The resulting solid was suspended
in saturated sodium bicarbonate solution (5 mL) before being
collected by filtration and dried under vacuum to give 4-(6-
chloropyrimidin-4-yl)morpholine (1.04 g, 78% yield) as a pale
brown solid. MS (ES+) m/z (M + 1) 200/202. This material was
then taken on as described in general procedure A above to give the
title compound 12. MS (ES+) m/z (M + 1) 210; 1H NMR (500 MHz,
1
DMSO) H NMR (500 MHz, DMSO-d6) δ 8.75 (s, 1H), 7.53 (s,
1H), 3.90 (s, 4H), 3.75 (dt, J = 4.72, 33.27 Hz, 4H). 13C NMR (126
MHz, DMSO) 161.79, 161.72, 153.15, 145.70, 103.95, 65.73, 45.38.
HRMS (ES+) m/z 210.0869 (210.0879 Calcd for C9H11N3O3 M + H).
6-[(3,4-Dichlorophenyl)amino]pyrimidine-4-carboxylic Acid (14).
3,4-Dichloroaniline (1.2 g, 7.8 mmol) was added in one portion to a
suspension of 4,6-dichloropyrimidine in isopropanol (7 mL) in a
pressure tube. The tube was sealed, heated to 80 °C, and stirred at this
temperature for 2 h. After this time, the tube was cooled to room
temperature and the resulting solid was collected by filtration and
dried under vacuum to give 6-chloro-N-(3,4-dichlorophenyl)-
pyrimidin-4-amine (0.8 g, 44% yield) as a white solid. MS (ES+) m/
z (M + 1) 275. This material was then taken on as described in general
procedure A above to give the title compound 14. MS (ES+) m/z (M
3-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]-4,5-dihydro-1,2,4-oxa-
diazol-5-one (35). A solution of 30 (2.6 g, 7.9 mmol), hydroxylamine
hydrochloride (0.58 g, 8.3 mmol), ethanol (50 mL), and
diisopropylethylamine (1.45 mL, 8.33 mmol) was stirred at 70 °C
under a nitrogen atmosphere for 2.5 h. After this time, the mixture was
concentrated and the resulting residue was washed with water (15 mL)
and ether (7 mL), before being air-dried for 30 min, and then dried in
vacuo to give 6-(3,4-dichlorophenyl)-N′-hydroxypyrimidine-4-carbox-
imidamide (2.1 g, 75% yield) as a white solid. MS (ES+) m/z (M + 1)
283.
1
+ 1) 284; H NMR (500 MHz, DMSO) 11.17 (br s, 1H), 8.85 (s,
1H), 8.26 (br s, 1H), 7.72 (d, 1H), 7.49−7.65 (m, 2H). 13C NMR
(126 MHz, DMSO) 163.67, 161.40, 156.54, 150.07, 139.04, 131.02,
130.72, 125.03, 121.42, 120.41, 108.72. HRMS (ES+) m/z 283.999
(283.9994 Calcd for C11H7Cl2N3O2 M + H).
6-(3,4-Dichlorophenoxy)pyrimidine-4-carboxylic Acid (13). Potas-
sium tert-butoxide (0.51 g, 3.0 mmol) in tert-butanol (5 mL) was
added dropwise over 5 min to a stirred solution of 3,4-dichlorophenol
(5 mL) in tert-butanol (5 mL). After 5 min, 4,6-dichloropyrimidine
(0.5 g, 3.0 mmol) was added in one portion and the resulting reaction
mixture was stirred at 35 °C for 2 h. After this time, the reaction was
quenched by the addition of water (20 mL) and the resulting mixture
extracted with diethyl ether (2 × 25 mL). The combined organic
extracts were dried (MgSO4), filtered, and concentrated. The resulting
residue was triturated with heptane (5 mL) to give 4-chloro-6-(3,4-
A mixture of 6-(3,4-dichlorophenyl)-N′-hydroxypyrimidine-4-car-
boximidamide (0.71 g, 2.5 mmol), CDI (0.6 g, 3.7 mmol), DBU (0.48
mL, 3.2 mmol), and 1,4-dioxane (12 mL) was stirred at 105 °C under
a nitrogen atmopshere for 4 h. After this time, the reaction mixture was
concentrated and treated with water (100 mL) and 6 M hydrochloric
acid (50 mL) before being extracted with ethyl acetate (2 × 150 mL).
The combined organic extracts were dried (Na2SO4), filtered, and
concentrated. The resulting residue was purified by preparative HPLC
to give the title compound (0.12 g, 13% yield) as a white powder. MS
1
(ES+) m/z (M + 1) 309; H NMR (500 MHz, DMSO) 13.64 (br s,
T
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 9.44−9.52 (m, 1H), 8.60−8.67 (m, 1H), 8.53−8.59 (m, 1H),
8.25−8.35 (m, 1H), 7.84−7.92 (m, 1H). 13C NMR (126 MHz,
DMSO) 162.18, 159.58, 159.15, 156.77, 151.34, 135.64, 134.74,
132.29, 131.47, 129.14, 127.41, 113.77. HRMS (ES+) m/z 308.9935
(308.9946 Calcd for C12H6Cl2N4O2 M + H).
precipitate was collected by filtration and dried under vacuum to
give the title compound (0.4 g, 76% yield) as a white solid. MS (ES+)
m/z (M + 1) 240; 1H NMR (500 MHz, DMSO) 8.45 (s, 1H), 8.20 (d,
J = 2.05 Hz, 1H), 7.94 (dd, J = 8.43, 1.97 Hz, 1H), 7.76 (d, J = 8.35
Hz, 1H), 7.02 (br s, 2H), 6.94 (s, 1H). 13C NMR (63 MHz, DMSO)
164.48, 158.75, 158.23, 137.90, 132.61, 131.72, 131.07, 128.16, 126.38,
100.25. HRMS (ES+) m/z 240.0087 (240.0095 Calcd for C10H7Cl2N3
M + H).
4-(3,4-Dichlorophenyl)-6-(1,2,4-oxadiazol-3-yl)pyrimidine (36). A
mixture of 6-(3,4-dichlorophenyl)-N′-hydroxypyrimidine-4-carboximi-
damide (0.12 g, 0.42 mmol), trimethyl orthoformate (3 mL), and
concentrated HCl (3 drops) was stirred at 100 °C for 1 h. After this
time, the reaction mixture was cooled to room temperature and the
resulting precipitate was collected by filtration and washed with
heptane (5 mL) before being dried under vacuum to give the title
compound (0.09 g, 74% yield) as a pale pink solid. MS (ES+) m/z (M
General Method D. N-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]-
benzenesulfonamide (38). Sodium hydride (0.3 g, 8.0 mmol) was
added portionwise to a stirred solution of 37 (0.61 g, 3.0 mmol) in
dioxane (10 mL), and the mixture was stirred at room temperature
under a nitrogen atmosphere for 1 h. After this time, benzenesulfonyl
chloride (0.49 g, 3.0 mmol) was added dropwise and stirring was
continued for 5 h. After this time, the mixture was partitioned between
ethyl acetate (20 mL) and water (5 mL). The organic layer was
separated, dried (MgSO4), filtered, and concentrated. The resulting
residue was triturated with diethyl ether, and the solid precipitate was
collected by filtration, washed with ether, and dried under vacuum to
give the title compound (0.17 g, 18% yield) as a white solid. MS (ES+)
1
+ 1) 293; H NMR (500 MHz, CDCl3) 9.44−9.51 (m, 1H), 8.94−
9.00 (m, 1H), 8.46−8.52 (m, 1H), 8.36 (m, 1H), 8.02−8.10 (m, 1H),
7.62−7.68 (m, 1H). 13C NMR (126 MHz, DMSO) 168.54, 166.03,
162.45, 159.44, 153.97, 135.96, 134.58, 132.26, 131.45, 129.22, 127.53,
115.80. HRMS (ES+) m/z 293.0011 (292.9997 Calcd for
C12H6Cl2N4O M + H).
4-(3,4-Dichlorophenyl)-6-(1H-1,2,3,4-tetrazol-5-yl)pyrimidine
(31). To a stirred solution of 30 (1.0 g, 4.0 mmol) in toluene (50 mL)
were added azido(trimethyl)silane (1.1 mL, 8.0 mmol) and dibutyl-
(oxo)stannane (0.1 g, 0.4 mmol) sequentially. The mixture was heated
to 115 °C and stirred at this temperature for 24 h under a nitrogen
atmosphere. After this time, the mixture was cooled to room
temperature and the mixture was concentrated. The resulting residue
was partitioned between ethyl acetate (200 mL) and saturated sodium
bicarbonate (200 mL). The aqueous layer was removed and acidifed to
pH 2 with HCl (6 M solution), and the resulting precipitate was
collected by filtration and washed with water (10 mL) before being
dried in a vacuum over overnight. The crude solid was then purified by
prep HPLC to give the title compound (0.33 g, 28% yield) as a white
solid. MS (ES+) m/z (M + 1) 293/295; 1H NMR (500 MHz, DMSO)
9.47 (s, 1H), 8.80 (s, 1H), 8.55 (d, J = 1.95 Hz, 1H), 8.30 (dd, J =
1.99, 8.47 Hz, 1H), 7.83 (d, J = 8.46 Hz, 1H). 13C NMR (126 MHz,
DMSO) 162.40, 159.29, 154.35, 152.27, 135.87, 134.62, 132.27,
131.43, 129.17, 127.47, 114.78. HRMS (ES+) m/z 293.0093 (293.0109
Calcd for C11H6Cl2N6 M + H).
1
m/z (M + 1) 380; H NMR (500 MHz, DMSO) 12.26 (br s, 1H),
8.75 (br s, 1H), 8.19 (d, J = 1.73 Hz, 1H), 8.00 (d, J = 7.57 Hz, 2H),
7.94 (dd, J = 8.51, 1.89 Hz, 1H), 7.82 (d, J = 8.51 Hz, 1H), 7.63−7.71
(m, 1H), 7.57−7.63 (m, 2H), 7.49 (br s, 1H). 13C NMR (63 MHz,
DMSO) 166.32, 158.32, 157.03, 146.16, 138.77, 131.98, 131.58,
130.97, 129.81, 127.91, 126.48, 126.16, 106.33. HRMS (ES+) m/z
380.0009 (380.0027 Calcd for C16H11Cl2N3O2S M + H).
N-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]methanesulfonamide
(39). This material was synthesized according to general procedure D
1
as described above. MS (ES+) m/z (M + 1) 318/320; H NMR (250
MHz, DMSO) 11.6 (br s, 1H), 8.89 (s, 1H), 8.25 (d, J = 1.63 Hz, 1H),
7.99 (dd, J = 1.85, 8.42 Hz, 1H), 7.82 (d, J = 8.43 Hz, 1H), 7.41 (s,
1H), 3.40 (s, 3H). 13C NMR (63 MHz, DMSO) 160.64, 159.01,
136.66, 133.79, 132.03, 131.40, 128.63, 126.94, 103.88, 41.81. HRMS
(ES+) m/z 317.9863 (317.9871 Calcd for C11H9Cl2N3O2S M + H).
N-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]acetamide (40). This
material was synthesized according to general procedure D as
1
described above. MS (ES+) m/z (M + 1) 282/284; H NMR (500
MHz, DMSO) 11.08 (s, 1H), 8.96 (d, J = 1.10 Hz, 1H), 8.55 (d, J =
0.95 Hz, 1H), 8.26 (d, J = 2.05 Hz, 1H), 8.01 (dd, J = 8.43, 2.13 Hz,
1H), 7.83 (d, J = 8.51 Hz, 1H), 2.18 (s, 3H). 13C NMR (63 MHz,
DMSO) 170.98, 161.40, 159.06, 158.55, 137.08, 133.67, 132.04,
131.42, 128.53, 126.88, 104.96, 24.27. HRMS (ES+) m/z 282.0213
(282.0201 Calcd for C12H9Cl2N3O M + H).
4-(3,4-Dichlorophenyl)-6-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)-
pyrimidine (32) and 4-(3,4-Dichlorophenyl)-6-(2-methyl-2H-1,2,3,4-
tetrazol-5-yl)pyrimidine (33). Sodium hydride (60%, 0.03 g, 0.7
mmol) was added in one portion to a cooled (0 °C), stirred solution
of 31 (0.2 g, 0.7 mmol) in DMF, and the reaction mixture was stirred
under a nitrogen atmosphere for 15 min. After this time, methyl iodide
(0.08 mL, 1.4 mmol) was added in one portion, the mixture was
allowed to warm to room temperature, and stirring was continued for a
further 15 h. After this time, the reaction was quenched by the addition
of water (10 mL) before being extracted with ethyl acetate (3 × 25
mL). The combined organic extracts were dried (MgSO4), filtered,
and concentrated. The resulting residue was then purified by flash
column chromatography (elution: 50% EtOAc, 50% heptane) to give
32 (0.048 g, 22% yield) as a beige powder MS (ES+) m/z (M + 1)
N-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]benzamide (41). This
material was synthesized according to general procedure D as
1
described above. MS (ES+) m/z (M + 1) 343/345; H NMR (500
MHz, DMSO) 11.41 (s, 1H), 9.20−8.89 (m, 1H), 8.82−8.63 (m, 1H),
8.29 (d, J = 2.00 Hz, 1H), 8.14−7.89 (m, 3H), 7.82 (d, J = 8.43 Hz,
1H), 7.64 (t, J = 7.38 Hz, 1H), 7.54 (t, J = 7.68 Hz, 2H). 13C NMR
(63 MHz, DMSO) 167.32, 161.48, 159.64, 158.51, 137.08, 133.73,
133.28, 132.65, 132.07, 131.42, 128.61, 128.52, 128.35, 126.98, 106.15.
HRMS (ES+) m/z 344.0347 (344.0357 Calcd for C17H11Cl2N3O M +
H).
1
307/309; H NMR (250 MHz, DMSO) 9.53 (d, J = 1.34 Hz, 1H),
8.87 (d, J = 1.36 Hz, 1H), 8.60 (d, J = 2.10 Hz, 1H), 8.34 (dd, J = 2.16,
8.50 Hz, 1H), 7.87 (d, J = 8.49 Hz, 1H), 4.48 (s, 3H); 13C NMR (63
MHz, DMSO) 162.32, 158.93, 152.86, 150.98, 135.83, 134.69, 132.32,
131.50, 129.24, 127.53, 116.16, 36.98; HRMS (ES+) m/z 307.0261
(307.0266 Calcd for C12H8Cl2N6 M + H) and 33 (0.048 g, 22% yield)
as a beige solid MS (ES+) m/z (M + 1) 307/309; 1H NMR (250 MHz,
DMSO) 9.45 (d, J = 1.34 Hz, 1H), 8.75 (d, J = 1.35 Hz, 1H), 8.58 (d, J
= 2.10 Hz, 1H), 8.33 (dd, J = 2.16, 8.49 Hz, 1H), 7.86 (d, J = 8.48 Hz,
1H), 4.53 (s, 3H). 13C NMR (63 MHz, DMSO) 162.61, 162.07,
159.47, 156.87, 154.51, 136.15, 134.42, 132.24, 131.43, 129.19, 127.49,
114.72; HRMS (ES+) m/z 307.0251 (307.0266 Calcd for C12H8Cl2N6
M + H).
6-(3,4-Dichlorophenyl)pyrimidin-4-amine (37). 29 (0.58 g, 2.6
mmol) was suspended in saturated aqueous ammonia solution (5 mL)
in a pressure tube. The tube was sealed and heated to 100 °C for 18 h.
After this time, the tube was cooled to room temperature and the
mixture was poured onto water (15 mL). The resulting solid
General Method E. 4-(3,4-Dichlorophenyl)-6-(1H-imidazol-1-yl)-
pyrimidine (42). Imidazole (0.16 g, 2.3 mmol) was added in one
portion to a solution of 29 (0.15 g, 0.58 mmol) in ethanol (2 mL) in a
microwave pressure vessel. The vessel was sealed and heated to 140 °C
for 1 h in a microwave. After this time, the reaction was cooled to
room temperature, the reaction mixture was concentrated, and the
resulting residue was triturated with 1:1 acetonitrile/water (5 mL).
The solid precipitate was collected by filtration and dried under
vacuum to give the title compound (0.04 g, 25% yield) as a white
1
powder. MS (ES+) m/z (M + 1) 291; H NMR (500 MHz, CDCl3)
9.03−9.07 (m, 1H), 8.48 (s, 1H), 8.17−8.21 (m, 1H), 7.87−7.92 (m,
1H), 7.67−7.71 (m, 1H), 7.53−7.59 (m, 2H), 7.21 (s, 1H). HRMS
(ES+) m/z 291.0195 (291.0204 Calcd for C13H8Cl2N4 M + H).
4-(3,4-Dichlorophenyl)-6-(1H-pyrazol-1-yl)pyrimidine (43). This
material was synthesized according to general procedure E as
1
described above. MS (ES+) m/z (M + 1) 291; H NMR (500 MHz,
U
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
CDCl3) 9.06 (s, 1H), 8.63−8.68 (m, 1H), 8.30−8.36 (m, 2H), 8.01−
8.06 (m, 1H), 7.85 (s, 1H), 7.59−7.64 (m, 1H), 6.57 (s, 1H). 13C
NMR (126 MHz, DMSO) 162.67, 158.69, 157.63, 144.28, 136.26,
134.21, 132.12, 131.30, 128.99, 128.21, 127.33, 109.83, 104.09. HRMS
(ES+) m/z 291.0203 (291.0204 Calcd for C13H8Cl2N4 M + H).
1-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]-2,3-dihydro-1H-imida-
zol-2-one (44). Sodium hydride (60%, 0.05 g, 1.2 mmol) was added in
one portion to a stirred solution of 29 (0.15 g, 0.58 mmol) in DMF (3
mL) in a microwave pressure vessel, and stirring was continued for 5
min. After this time, 1-methylimidazol-2-one (0.08 g, 0.87 mmol) was
added in one portion, the vessel was sealed and heated to 85 °C for 5
min in a microwave. After this time, the reaction was cooled to room
temperature and partitioned between DCM (10 mL) and saturated
sodium bicarbonate (10 mL). The organic layer was separated, dried
(MgSO4), filtered, and concentrated. The resulting residue was
purified by flash column chromatography (elution: 50% EtOAc, 50%
heptane) to give the title compound (0.1 g, 59% yield) as a light
pressure, and the solid precipitate was collected by filtration and
washed with water (10 mL) before being dried in a vacuum over
overnight to give the hydrochloride salt of compound 50 (0.54 g, 57%
1
yield) as a white solid. MS (ES+) m/z (M + 1) 268, 270. H NMR
(500 MHz, DMSO) 8.78 (d, J = 5.04 Hz, 1H), 8.31 (d, J = 1.10 Hz,
1H), 8.17 (d, J = 2.21 Hz, 1H), 8.00 (dd, J = 5.04, 1.73 Hz, 1H), 7.87
(m, J = 8.35, 2.21 Hz, 1H) 7.77−7.81 (m, 1H). 13C NMR (126 MHz,
DMSO) 166.07, 154.53, 150.83, 139.75, 138.45, 132.27, 131.90,
131.13, 128.50, 126.84, 122.40, 119.51. HRMS (ES+) m/z 267.9932
(267.9938 Calcd for C12H7Cl2NO2 M + H).
2-(3,4-Dichlorophenyl)pyridine-4-carboxylic Acid (49). This ma-
terial was synthesized according to general procedure F as described
above. MS (ES+) m/z (M + 1) 268, 270; 1H NMR (500 MHz, CDCl3)
13.85 (br s, 1H), 8.87 (d, J = 5.04 Hz, 1H), 8.33−8.44 (m, 2H), 8.14
(dd, J = 8.51, 1.89 Hz, 1H), 7.83 (d, J = 4.89 Hz, 1H), 7.77 (d, J = 8.35
Hz, 1H). 13C NMR (126 MHz, DMSO) 166.00, 154.46, 150.75,
139.74, 138.40, 132.19, 131.82, 131.06, 128.43, 126.78, 122.32, 119.44.
HRMS (ES+) 267.9933 (267.9932 Calcd for C12H7Cl2N2O2 M + H).
General Method G. 6-(7-Chlorobenzofuran-5-yl)pyrimidine-4-
carboxylic Acid (77). Potassium carbonate (13.3 g, 96.4 mmol) was
added portionwise to a stirred solution of 4-bromo-2-chlorophenol
(10.0 g, 48.2 mmol) and bromoacetaldehyde diethyl acetal (8.91 mL,
57.8 mmol) in DMF (60 mL), and the mixture was heated to 140 °C
and heated at this temperature under a nitrogen atmosphere for 3 h.
After this time, the reaction mixture was cooled to room temperature
and concentrated. The resulting residue was partitioned between ethyl
acetate (200 mL) and water (50 mL), and the organic layer was
separated, dried (MgSO4), filtered, and concentrated. The resulting
residue was purified using a Biotage Isolera (340 g silica column
eluting with a gradient from 0% DCM to 60% DCM/40% heptane) to
give 4-bromo-2-chloro-1-(2,2-diethoxyethoxy)benzene (14.8 g, 90%
yield) as a colorless oil. tR = 2.42 min m/z (ES+) (M + Na+) 347.
4-Bromo-2-chloro-1-(2,2-diethoxyethoxy)benzene (8.47 g, 26.2
mmol) was added portionwise as a solution in toluene (36 mL) to
polyphosphonic acid (17.0 g, 207.3 mmol) at 0 °C. The resulting
suspension was allowed to warm to room temperature before being
heated to reflux and stirred for 1 h. After this time, the mixture was
cooled to room temperature and partitioned between water (100 mL)
and ethyl acetate (300 mL). The resulting residue was partitioned
between ethyl acetate (300 mL) and water (50 mL), and the organic
layer was separated, dried (MgSO4), filtered, and concentrated. The
resulting residue was purified using a Biotage Isolera (340 g silica
column eluting with 100% heptane) to give 5-bromo-7-chlorobenzo-
furan (1.63 g, 26% yield) as a white solid. tR = 2.29 min m/z (ES+) (M
+ Na+) 254.
Potassium acetate (1.97 g, 31.5 mmol) was added in one portion to
a stirred solution of 5-bromo-7-chlorobenzofuran (2.43 g, 10.5 mmol)
and bis-pinacol borane (2.93 g, 11.5 mmol) in DMF (30 mL). The
mixture was degassed with nitrogen for 5 min, after which time
Pd(dppf)2Cl2 (0.26 g, 0.31 mmol) was added in one portion, and the
mixture was then heated to 80 °C and stirred at this temperature for
18 h under a nitrogen atmosphere. After this time, the reaction mixture
was cooled to room temperature and partitioned between ethyl acetate
(200 mL) and water (100 mL). The biphasic suspension was filtered
through glass fiber filter paper, and the organic layer was separated and
washed sequentially with water (3 × 100 mL) before being dried
(MgSO4), filtered, and concentrated. The resulting residue was
purified purified using a Biotage Isolera (100g silica column eluting
with 100% heptane to 50% DCM/50% heptane) to give 7-chloro-5-
(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)benzofuran (2.23 g, 73%
yield) as a white solid. tR = 2.53 min m/z (ES+) (M + H+) 279.
Tripotassium phosphate (2.76 g, 12.0 mmol) was added in one
portion to a stirred solution of 7-chloro-5-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)benzofuran (2.23 g, 8.0 mmol) and methyl
6-chloropyrimidine-4-carboxylate (1.7 g, 9.6 mmol) in DMF (35 mL).
The mixture was degassed with nitrogen for 5 min, after which time
Pd(dppf)2Cl2 (0.33 g, 0.4 mmol) was added in one portion, the
mixture was then heated to 60 °C and stirred at this temperature for
16 h under a nitrogen atmosphere. After this time, the reaction mixture
was cooled to room temperature and partitioned between ethyl acetate
1
brown solid. MS (ES+) m/z (M + 1) 307/308; H NMR (500 MHz,
DMSO) 10.75 (br s, 1H), 9.09 (s, 1H), 8.90 (s, 1H), 8.29 (d, J = 1.98
Hz, 1H), 8.05 (dd, J = 1.98, 8.54 Hz, 1H), 7.86 (d, J = 8.54 Hz, 1H),
7.31 (d, J = 3.05 Hz, 1H), 6.77 (d, J = 3.20 Hz, 1H). HRMS (ES+) m/
z 307.0146 (307.0153 Calcd for C13H8Cl2N4O M + H).
1-[6-(3,4-Dichlorophenyl)pyrimidin-4-yl]-3-methyl-2,3-dihydro-
1H-imidazol-2-one (45). Sodium hydride (60%, 0.02 g, 0.8 mmol)
was added portionwise to a stirred solution of 1-[6-(3,4-
dichlorophenyl)pyrimidin-4-yl]-2,3-dihydro-1H-imidazol-2-one (0.1
g, 0.33 mmol) in DMF (3 mL), and the mixture was stirred at
room temperature under a nitrogen atmosphere for 15 min. After this
time, methyl iodide (0.04 mL, 0.65 mmol) was added in one portion
and stirring was continued for 15 h. After this time, the mixture was
partitioned between ethyl acetate (20 mL) and water (5 mL). The
organic layer was separated, dried (MgSO4), filtered, and concentrated.
The resulting residue was purified by flash column chromatography
(elution: 30% EtOAc, 70% heptane) to give the title compound (0.07
1
g, 72% yield) as a white solid. MS (ES+) m/z (M + 1) 321/323; H
NMR (500 MHz, DMSO) 9.09 (d, J = 0.92 Hz, 1H), 8.88 (d, J = 0.92
Hz, 1H), 8.28 (d, J = 2.14 Hz, 1H), 8.05 (dd, J = 2.14, 8.39 Hz, 1H),
7.86 (d, J = 8.54 Hz, 1H), 7.36 (d, J = 3.36 Hz, 1H), 6.89 (d, J = 3.20
Hz, 1H), 3.24 (s, 3H). 13C NMR (63 MHz, DMSO) 161.80, 158.61,
156.03, 151.40, 136.70, 134.00, 132.13, 131.53, 128.57, 126.95, 115.93,
105.71, 104.11, 29.98. HRMS (ES+) m/z 321.0291 (321.031 Calcd for
C14H10Cl2N4O M + H).
General Method F. 4-(3,4-Dichlorophenyl)pyridine-2-carboxylic
Acid (50). Tripotassium phosphate (2.45 g, 12.0 mmol) was added in
one portion to a stirred solution of 3,4-dichlorophenyl boronic acid
(1.47 g, 8.0 mmol) and methyl 4-bromopyridine-2-carboxylate (2.0 g,
9.0 mmol) in DMF (30 mL). The mixture was degassed with nitrogen
for 5 min, after which time Pd(dppf)2Cl2 (0.31 g, 0.38 mmol) was
added in one portion, and the mixture was then heated to 60 °C and
stirred at this temperature for 16 h under a nitrogen atmosphere. After
this time, the reaction mixture was cooled to room temperature and
partitioned between ethyl acetate (200 mL) and water (100 mL). The
organic layer was separated and washed sequentially with water (100
mL) and then brine (100 mL) before being dried (MgSO4), filtered,
and concentrated. The resulting brown solid was purified by flash
column chromatography (elution: 50% EtOAc, 50% heptane) to give
4-(3,4-dichlorophenyl)pyridine-2-carboxylic acid methyl ester (1.41g,
1
65% yield) as a white solid. MS (ES+) m/z (M + 1) 282, 284. H
NMR (500 MHz, DMSO) 8.77−8.83 (1 H, m), 8.33−8.39 (1 H, m),
8.18−8.22 (1 H, m), 8.02−8.09 (1 H, m), 7.86−7.93 (1 H, m), 7.78−
7.83 (1 H, m), 3.92 (3 H, s).
NaOH (2 M solution, 3.1 mL, 6.0 mmol) was added in one portion
to a stirred solution of 4-(3,4-dichlorophenyl)pyridine-2-carboxylic
acid methyl ester (0.87 g, 3.0 mmol) in THF (50 mL), and the mixture
was stirred at room temperature for 16 h. After this time, the resulting
precipitate was collected by filtration and washed with water (1 mL)
and DCM (20 mL) before being dried under vacuum. This solid was
then suspended in HCl (2 M solution, 60 mL) and acetonitrile (60
mL), heated to 80 °C until complete dissolution, and then cooled to
room temperature. The acetonitrile was removed under reduced
V
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(200 mL) and water (100 mL). The organic layer was separated and
washed sequentially with water (100 mL) and then brine (100 mL)
before being dried (MgSO4), filtered, and concentrated. The resulting
residue was purified purified using a Biotage Isolera (50 g silica column
eluting with 100% heptane to 20% ethyl acetate/50% heptane) to give
6-(7-chlorobenzofuran-5-yl)pyrimidine-4-carboxylic acid methyl ester
(0.37 g, 16% yield) as a white solid. tR = 2.01 min m/z (ES+) (M +
H+) 289, 291.
PD studies; methods for tissue distribution study; methods for
analysis of KYN, KYNA, 3-HK, and AA; methods for analysis of
QA; methods for microdialysis studies; methods for quantifi-
cation of KP metabolites in plasma and microdialysate samples;
methods for quantification of compound 75 in plasma and
microdialysate samples. This material is available free of charge
via the Internet at http://pubs.acs.org
NaOH (2 M solution, 3.2 mL, 2.3 mmol) was added in one portion
to a stirred solution of 6-(7-chlorobenzofuran-5-yl)pyrimidine-4-
carboxylic acid methyl ester (0.37 g, 1.3 mmol) in THF (8 mL),
and the mixture was stirred at room temperature for 16 h. After this
time, the resulting precipitate was collected by filtration, washed with
water (1 mL), and dried under vacuum to give compound 77 (0.06 g,
AUTHOR INFORMATION
Corresponding Author
*E-mail: Leticia.Toledo-Sherman@CHDIFoundation.org.
Notes
■
1
16% yield) as a white solid. MS (ES+) m/z (M + 1) 275/277. H
The authors declare no competing financial interest.
NMR (500 MHz, DMSO) 14.02 (br s, 1 H) 9.40 (s, 1 H) 8.65 (d, J =
1.26 Hz, 1 H) 8.60 (s, 1 H) 8.37 (d, J = 1.26 Hz, 1 H) 8.24 (d, J = 2.05
Hz, 1H) 7.21 (d, J = 2.05 Hz, 1 H). 13C NMR (63 MHz, d4-AcOH)
168.55, 167.72, 159.93, 157.85, 154.35, 149.27, 133.64, 131.62, 125.62,
121.78, 119.33, 118.30, 109.56. HRMS (ES+) m/z 275.0229 (275.0223
Calcd for C13H7ClN2O3 M + H).
ACKNOWLEDGMENTS
■
We thank Teri Buchanan and Tod Wehrman of Xenometrics
for help on the in-life portion of the PK study with compound
75. We also thank Helen Birch and Tania Mead, of BioFocus
DPI Ltd., who contributed to the bioanaltyical work for the PK
study on Bcrp(−/−), Mdr1a/b(−/−), and Mdr1a/b(−/
−)/Bcrp(−/−) mice.
General Method H. N-(Benzenesulfonyl)-6-(3,4-dichlorophenyl)-
pyrimidine-4-carboxamide (51). Oxalyl chloride (0.34 mL, 4.23
mmol) was added dropwise to a stirred solution of 7 (0.38 g, 1.41
mmol) in DCM (15 mL) containing 1 drop of DMF, and the reaction
mixture was stirred at room temperature for 30 min. After this time,
benzenesulfonamide (0.33 g, 2.1 mmol) was added followed by
triethylamine (0.58 mL, 4.23 mmol) and the resulting solution was
stirred at room temperature for a further 1 h. The resulting mixture
was concentrated, diluted with DCM (30 mL), and washed
consecutively with water (20 mL), saturated sodium bicarbonate (20
mL), citric acid (20 mL, 2 M solution), and brine (20 mL). The
organic layer was dried (Na2SO4), filtered, and concentrated. The
resulting residue was triturated with 1:1 MeCN/H2O and the solid
precipitate collected by filtration before being further purified by flash
column chromatography (elution: 10% ethyl acetate, 90% heptane) to
give compound 51 (0.06g, 10% yield) as a white solid. MS (ES+) m/z
(M + 1) 408. 1H NMR (500 MHz, CDCl3) 10.32 (br s, 1H), 9.30 (s,
1H), 8.39 (s, 1H), 8.29 (s, 1H), 8.24−8.18 (m, 2H), 7.99−7.77 (m,
1H), 7.67−7.72 (m, 1H), 7.57−7.63 (m, 3H). 13C NMR (63 MHz,
DMSO) 163.99, 162.46, 158.53, 140.45, 136.05, 134.46, 133.19,
132.21, 131.40, 129.17, 128.91, 127.65, 127.46, 115.40. HRMS (ES+)
m/z 407.9971 (407.9976 Calcd for C17H11Cl2N3O3S M + H).
Pharmacokinetics. The pharmacokinetics of 75 were determined
following a single iv dose of 6.2 mg equiv/kg or oral gavage at 12 mg
equiv/kg administration to nonfasted male Sprague−Dawley rats
(body weight 300−370 g, Hilltop Laboratories, Scottsdale, PA).
Compound was formulation for iv administration as a 1.24 mg equiv/
mL solution in DMSO:PEG 400: Water (10:40:50 v/v/v) and dosed
via the tail vein. A 1.20 mg equiv/mL solution in 10% hydroxypropyl-
β-cyclodextrin in water was dosed orally by gavage. Doses were
administered to each animal on a volumetric basis, calculated from the
animal’s nonfasting weight. Animals were anesthetized using
isoflurance inhalation and terminally bled (N = 3) via abdominal
aorta or cardiac puncture at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h. Blood
was treated with sodium heparin and plasma prepared by
centrifugation. Tissues were harvested, rinsed with saline, and snap-
frozen in liquid nitrogen. All samples were frozen at −70 °C until
analyzed by LC-MS.
ABBREVIATIONS USED
■
SAR, structure−activity relationship; ADME, absorption
distribution metabolism excretion; NAD, nicotinamide adenine
nucleotide; PK, pharmacokinetics; PD, pharmacodynamics;
NBS, N-bromosuccinimide; AIBN, azobis(isobutyronitrile);
LCMS, liquid chromatography mass spectrometry; CHO,
Chinese hamster ovary; BCRP, breast cancer resistance protein;
MDCK, Madin−Darby canine kidney; MDR1, multidrug
resistance protein 1; AUCN, area under the curve normalized;
CSF, cerebrospinal fluid; EER, effective efflux ratio; KYN,
kynurenine; KYNA, kynurenic acid; 3-HK, 3-hydroxykynur-
enice acid; AA, anthranilic acid; QA, quinolinic acid
REFERENCES
■
(1) Reiner, A.; Dragatsis, I.; Dietrich, P. Genetics and neuropathology
of Huntington’s disease. Int. Rev. Neurobiol. 2011, 98, 325−372.
(2) Delmaire, C.; Dumas, E. M.; Sharman, M. A.; van den Bogaard, S.
J.; Valabregue, R.; Jauffret, C.; Justo, D.; Reilmann, R.; Stout, J. C.;
́
Craufurd, D.; Tabrizi, S. J.; Roos, R. A.; Durr, A.; Lehericy, S. The
structural correlates of functional deficits in early Huntington’s
Disease. Hum. Brain Mapping 2013, 34 (9), 2141−2153.
(3) Poon, L. H.; Kang, G. A.; Lee, A. J. Role of tetrabenazine for
Huntington’s disease-associated chorea. Ann. Pharmacother. 2010, 44
(6), 1080−1089.
(4) Schwarcz, R.; Bruno, J. P.; Muchowski, J. P.; Wu, H.-Q.
Kynurenines in the mammalian brain: When physiology meets
pathology. Nat. Rev. Neurosci. 2012, 13 (7), 465−477.
(5) Vecsei, L.; Szalardy, L.; Fulop, F.; Toldi, J. Kynurenines in the
́ ́
̈
̈
CNS: Recent advances and new questions. Nat. Rev. Drug Discovery
2013, 12 (1), 64−82.
(6) Mandi, Y.; Vecsei, L. The kynurenine systems and immunor-
egulation. J. Neural. Transm. 2012, 119 (2), 197−209.
(7) Belenky, P.; Bogan, K. L.; Brenner, C. NAD+ metabolism in
health and disease. Trends Biochem. Sci. 2006, 32 (1), 12−19.
(8) Chen, Y.; Guillemin, G. J. Kynurenine Pathway Metabolites in
Humans: Disease and Healthy States. Int. J. Tryptophan Res. 2009, 2,
1−19.
(9) Schwarcz, R.; Guidetti, P.; Sathyasaikumar, K. P.; Muchowski, P.
J. Of mice, rats and men: Revisiting the quinolinic acid hypothesis of
Huntington’s disease. Prog. Neurobiol. 2010, 90, 230−245.
(10) Guidetti, P.; Luthi-Carter, R. E.; Augood, S. J.; Schwarcz, R.
Neostriatal and cortical quinolinate levels are increased in early grade
Huntington’s disease. Neurobiol. Dis. 2004, 17, 455−461.
ASSOCIATED CONTENT
* Supporting Information
■
S
Methods for permeability and efflux studies; methods for in
vivo studies in efflux transporter knock out mouse model;
methods for sample preparation and LC-MS/MS analysis
conditions; methods for plasma extraction; methods for tissue
homogenate preparation; methods for LC-MS/MS method-
ology; methods for pharmacokinetic analysis; methods for
pharmacodynamic in vivo studies; methods for terminal PK/
W
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(11) Guidetti, P.; Bates, G. P.; Graham, R. K.; Hayden, M. R.; Leavitt,
B. R.; MacDonald, M. E.; Slow, E. J.; Wheeler, V. C.; Woodman, B.;
Schwarcz, R. Elevated brain 3-hydroxykynurenine and quinolinate
levels in Huntington disease mice. Neurobiol. Dis. 2006, 23 (1), 190−
197.
(12) Forrest, C. M.; Mackay, G. M.; Stoy, N.; Spiden, S. L.; Taylor,
R.; Stone, T. W.; Darlington, L. G. Blood levels of kynurenines,
interleukin-23 and soluble human leucocyte antigen-G at different
stages of Huntington’s disease. J. Neurochem. 2010, 112, 112−122.
(13) Thevandavakkam, M. A.; Schwarcz, R.; Muchowski, P. J.;
Giorgini, F. Targeting kynurenine 3-monooxygenase (KMO):
implications for therapy in Huntington’s disease. CNS Neurol. Disord.:
Drug Targets 2010, 9, 791−800.
neuroprotector kynurenic acid increases neuronal cell survival through
neprilysin induction. Neuropharmacology 2013, 70, 254−260.
(29) Stone, T. W. Development and therapeutic potential of
kynurenic acid and kynurenine derivatives for neuroprotection. Trends
Pharmacol. Sci. 2000, 21 (4), 149−154.
(30) Stone, T. W.; Darlington, L. G. Endogenous kynurenines as
targets for drug discovery and development. Nat. Rev. Drug. Discovery
2002, 1 (8), 609−620.
(31) Storey, E.; Cipolloni, P. B.; Ferrante, R. J.; Kowall, N. W.; Beal,
M. F. Movement disorder following excitotoxin lesions in primates.
Neuroreport. 1994, 5 (10), 1259−1261.
(32) Schwarcz, R.; Whetsell, W. O., Jr.; Mangano, R. M. Quinolinic
acid: An endogenous metabolite that produces axon-sparing lesions in
rat brain. Science 1983, 219 (4582), 316−318.
(33) Beal, M. F.; Ferrante, R. J.; Swartz, K. J.; Kowall, N. W. Chronic
quinolinic acid lesions in rats closely resemble Huntington’s disease. J.
Neurosci. 1991, 11 (6), 1649−1659.
(14) Giorgini, F.; Guidetti, P.; Nguyen, Q.; Bennett, S. C.;
Muchowski, P. J. A genomic screen in yeast implicates kynurenine
3-monooxygenase as a therapeutic target for Huntington disease. Nat.
Genet. 2005, 37 (5), 526−531.
(34) Okuda, S.; Nishiyama, N.; Saito, H.; Katsuki, H. Hydrogen
peroxide-mediated neuronal cell death induced by an endogenous
neurotoxin, 3-hydroxykynurenine. Proc. Natl. Acad. Sci. U. S. A. 1996,
93, 12553−12558.
(15) Campesan, S.; Green, E. W.; Breda, C.; Sathyasaikumar, K. V.;
Muchowski, P. J.; Schwarcz, R.; Kyriacou, C. P.; Giorgini, F. The
kynurenine pathway modulates neurodegeneration in a Drosophila
model of Huntington’s disease. Curr. Biol. 2011, 21 (11), 961−966.
(16) Pellicciari, R.; Amori, L.; Costantino, G.; Giordani, A.;
Macchiarulo, A.; Mattoli, L.; Pevarello, P.; Speciale, C.; Varasi, M.
Modulation of the kynurine pathway of tryptophan metabolism in
search for neuroprotective agents. Focus on kynurenine-3-hydroxylase.
Adv. Exp. Med. Biol. 2003, 527, 621−628.
(35) Stone, T. W.; Mackay, G. M.; Forrest, C. M.; Catherine, J.;
Clark, C. J.; Darlington, L. G. Tryptophan Metabolites and Brain
Disorders. Clin. Chem. Lab. Med. 2003, 41 (7), 852−859.
́ ́
(36) Majlath, Z.; Tajti, J.; Vecsei, L. Kynurenines and other novel
therapeutic strategies in the treatment of dementia. Ther. Adv. Neurol.
Disord. 2013, 6 (6), 386−397.
(37) Parsons, C. G.; Danysz, W.; Quack, G.; Hartmann, S.; Lorenz,
B.; Wollenburg, C.; Baran, L.; Przegalinski, E.; Kostowski, W.;
Krzascik, P.; Chizh, B.; Headley, P. M. Novel systemically active
antagonists of the glycine site of the N-methyl-d-aspartate receptor:
Electrophysiological, biochemical and behavioral characterization. J.
Pharmacol. Exp. Ther. 1997, 283, 1264−1275.
(38) Sapko, M. T.; Guidetti, P.; Yu, P.; Tagle, D. A.; Pellicciari, R.;
Schwarcz, R. Endogenous kynurenate controls the vulnerability of
striatal neurons to quinolinate: Implications for Huntington’s disease.
Exp. Neurol. 2006, 197 (1), 31−40.
(17) Amori, L.; Guidetti, P.; Pellicciari, R.; Kajii, Y.; Schwarcz, R. On
the relationship between the two branches of the kynurenine pathway
in the rat brain in vivo. J. Neurochem. 2009, 109 (2), 316−325.
(18) Heyes, M. P.; Chen, C. Y.; Major, E. O.; Saito, K. Different
kynurenine pathway enzymes limit quinolinic acid formation by
various human cell types. Biochem. J. 1997, 326 (2), 351−356.
(19) Guillemin, G. J.; Smith, D. G.; Smythe, G. A.; Armati, P. J.;
Brew, B. J. Expression of the kynurenine pathway enzymes in human
microglia and macrophages. Adv. Exp. Med. Biol. 2003, 527, 105−112.
(20) Rzeski, W.; Kocki, T.; Dybel, A.; Wejksza, K.; Zdzisin
́
ska, B.;
Kandefer-Szerszen, M.; Turski, W. A.; Okuno, E.; Albrecht, J.
́
(39) Poeggeler, B.; Rassoulpour, A.; Wu, H. Q.; Guidetti, P.; Roberts,
R. C.; Schwarcz, R. Dopamine receptor activation reveals a novel,
kynurenate-sensitive component of striatal N-methyl-D-aspartate
neurotoxicity. Neuroscience 2007, 148 (1), 188−197.
(40) Chiarugi, A.; Carpenedo, R.; Moroni, F. Kynurenine disposition
in blood and brain of mice: Effects of selective inhibitors of kynurenine
hydroxylase and of kynureninase. J. Neurochem. 1996, 67 (2), 692−
698.
(41) Speciale, C.; Wu, H. Q.; Cini, M.; Marconi, M.; Varasi, M.;
Schwarcz, R. (R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a
large and persistent increase in brain kynurenic acid levels in rats. Eur.
J. Pharmacol. 1996, 315 (3), 263−267.
Demonstration of kynurenine aminotransferases I and II and
characterization of kynurenic acid synthesis in cultured cerebral
cortical neurons. J. Neurosci. Res. 2005, 80 (5), 677−682.
(21) Schwarcz, R.; Pellicciari, R. Manipulation of brain kynurenines:
glial targets, neuronal effects, and clinical opportunities. J. Pharmacol.
Exp. Ther. 2002, 303, 1−10.
(22) Guillemin, G. J.; Cullen, K. M.; Lim, C. K.; Smythe, G. A.;
Garner, B.; Kapoor, V.; Takikawa, O.; Brew, B. J. Characterization of
the kynurenine pathway in human neurons. J. Neurosci. 2007, 27,
12884−12892.
(23) Schwarcz, R.; Bruno, J. P.; Muchowski, P. J.; Wu, H. Q.
Kynurenines in the mammalian brain: When physiology meets
pathology. Nat. Rev. Neurosci. 2012, 13, 465−477.
(24) Fukui, S.; Schwarcz, R.; Rapoport, S. I.; Takada, Y.; Smith, Q. R.
Blood-Brain Barrier Transport of Kynurenines: Implications for Brain
Synthesis and Metabolism. J. Neurochem. 1991, 56 (6), 2007−2017.
(25) Zwilling, D.; Huang, S. Y.; Sathyasaikumar, K. V.; Notarangelo,
F. M.; Guidetti, P.; Wu, H. Q.; Lee, J.; Truong, J.; Andrews-Zwilling,
Y.; Hsieh, E. W.; Louie, J. Y.; Wu, T.; Scearce-Levie, K.; Patrick, C.;
Adame, A.; Giorgini, F.; Moussaoui, S.; Laue, G.; Rassoulpour, A.; Flik,
G.; Huang, Y.; Muchowski, J. M.; Masliah, E.; Schwarcz, R.;
Muchowski, P. J. Kynurenine 3-monooxygenase inhibition in blood
ameliorates neurodegeneration. Cell. 2011, 145 (6), 863−874.
(26) Chiarugi, A.; Carpenedo, R.; Moroni, F. Kynurenine Disposition
in Blood and Brain of Mice: Effects of Selective Inhibitors of
Kynurenine Hydroxylase and of Kynureninase. J. Neurochem. 1996, 67
(2), 692−698.
(42) Sapko, M. T.; Guidetti, P.; Yu, P.; Tagle, D. A.; Pellicciari, R.;
Schwarcz, R. Endogenous kynurenate controls the vulnerability of
striatal neurons to quinolinate: Implications for Huntington’s disease.
Exp. Neurol. 2006, 197, 31−40.
(43) Rover, S.; Cesura, A. M.; Huguenin, P.; Kettler, R.; Szente, A.
̈
Synthesis and Biochemical Evaluation of N-(4-Phenylthiazol-2-yl)-
benzenesulfonamides as High-Affinity Inhibitors of Kynurenine 3-
Hydroxylase. J. Med. Chem. 1997, 40, 4378−4385.
(44) Moroni, F.; Cozzi, A.; Peruginelli, F.; Carpenedo, R.; Pellegrini-
Giampietro, D. E. Neuroprotective effects of kynurenine-3-hydroxylase
inhibitors in models of brain ischemia. Adv. Exp. Med. Biol. 1999, 467,
199−206.
(45) Cozzi, A.; Carpenedo, R.; Moroni, F. Kynurenine hydroxylase
inhibitors reduce ischemic brain damage: Studies with (m-nitro-
benzoyl)-alanine (mNBA) and 3,4-dimethoxy-[N-4-(nitrophenyl)-
thiazol-2yl]-benzenesulfonamide (Ro 61-8048) in models of focal or
global brain ischemia. J. Cereb. Blood Flow Metab. 1999, 19 (7), 771−
777.
(27) Saito, K.; Markey, S. P.; Heyes, M. P. Effects of immune
activation on quinolinic acid and neuroactive kynurenines in the
mouse. Neuroscience 1992, 51, 25−39.
(28) Klein, C.; Patte-Mensah, C.; Taleb, O.; Bourguignon, J.-J.;
Schmitt, M.; Bihel, F.; Maitre, M.; Mensah-Nyagan, A. G. The
́
(46) Samadi, P.; Gregoire, L.; Rassoulpour, A.; Guidetti, P.; Izzo, E.;
Schwarcz, R.; Bed
́
ard, P. J. Effect of kynurenine 3-hydroxylase
X
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
inhibition on the dyskinetic and antiparkinsonian responses to
levodopa in Parkinsonian monkeys. Mov. Disord. 2005, 20 (7), 792−
802.
(47) Beconi, M. G.; Yates, D.; Lyons, K.; Matthews, K.; Clifton, S.;
Mead, T.; Michael Prime, M.; Winkler, D.; O’Connell, C.; Walter, D.;
Toledo-Sherman, L. M.; Munoz-Sanjuan, I.; Dominguez, C.
Metabolism and Pharmacokinetics of JM6 in Mice: JM6 Is Not a
Prodrug for Ro-61-8048. Drug Metab. Dispos. 2012, 40 (12), 2297−
2306.
(48) Courtney, S. M.; Prime, M.; Mitchell, W.; Brown, C. J.; De
Aguiar Pena, P. C.; Johnson, P.; Dominguez, C.; Toledo-Sherman, L.
M.; Munoz, I. Kynurenine-3-Monooxygenase Inhibitors, Pharmaceut-
ical Compositions and Methods of use Thereof. International Patent
WO2013/033085, Mar 7, 2013.
(49) Dominguez, C.; Toledo-Sherman, L. M.; Courtney, S. M.;
Prime, M.; Mitchell, W.; Brown, C. J.; De Aguiar Pena, P. C.; Johnson,
P. Certain Kynurenine-3-Monooxygenase Inhibitors, Pharmaceutical
Compositions and Methods of use Thereof. International Patent
WO2013/016488, Jan 31, 2013.
(64) Amaral, M.; Levy, C.; Heyes, D. J.; Lafite, P.; Outeiro, T. F.;
Giorgini, F.; Leys, D.; Scrutton, N. S. Structural basis of kynurenine 3-
monooxygenase inhibition. Nature 2013, 496 (7445), 382−385.
́ ́ ́
(65) Krajcsi, P.; Jani, M.; Toth, B.; Erdo, F.; Kis, E.; Beery, E.; Sziraki,
I. Efflux transporters in the blood-brain interfaces-in vitro and in vivo
methods and correlations. Expert Opin. Drug Metab. Toxicol. 2012, 8
(4), 419−431.
(66) Jauch, D. A.; Sethy, V. H.; Weick, B. G.; Chase, T. N.; Schwarcz,
R. Intravenous administration of L-kynurenine to rhesus monkeys:
Effect on quinolinate and kynurenate levels in serum and cerebrospinal
fluid. Neuropharmacology 1993, 32 (5), 467−472.
(67) Alexander, K. S.; Wu, H. Q.; Schwarcz, R.; Bruno, J. P. Acute
elevations of brain kynurenic acid impair cognitive flexibility:
normalization by the alpha7 positive modulator galantamine.
Psychopharmacology 2012, 220 (3), 627−637.
(68) Westerink, B. H. C.; Cremers, T. I. F. H., Eds. Handbook of
Microdialyis − Methods, Applications and Clinical Aspects; Academic
Press, Elsevier: London, UK. 2007.
(50) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling
Reactions of Organoboron Compounds. Chem. Rev. 1995, 95 (7),
2457−2483.
(51) Albaneze-Walker, J.; Bazaral, C.; Leavey, T.; Dormer, P. G.;
Murry, J. A. Improved Carbonylation of Heterocyclic Chlorides and
Electronically Challenging Aryl Bromides. Org. Lett. 2004, 6 (13),
2097−2100.
(52) van der Westhuyzen, C. W.; Rousseau, A. L.; Parkinson, C. J.
Effect of Substituent Structure on Pyrimidine Electrophilic Sub-
stitution. Tetrahedron 2007, 63 (25), 5394−5405.
(53) Hayashi, T.; Konishi, M.; Kobori, Y.; Kumada, M.; Higuchi, T.
Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II): An ef-
fective catalyst for cross-coupling of secondary and primary alkyl
Grignard and alkylzinc reagents with organic halides. J. Am. Chem. Soc.
1984, 106 (1), 158−163.
(54) Pearson, R. E.; Martin, J. C. The Mechanism of Benzylic
Bromination with N-Bromosuccinimide. J. Am. Chem. Soc. 1963, 85
(3), 354−355.
(55) Bernard, A. M.; Ghiani, M. R.; Piras, P. P.; Rivoldini, A.
Dealkylation of Activated Alkyl Aryl Ethers Using Lithium Chloride in
Dimethylformamide. Synthesis 1989, 4, 287−289.
́ ́ ́
(56) Dolakova, P.; Masojídkova, M.; Holy, A. Efficient Synthesis of
Pyrimidinecarbonitriles and Their Derivatives. Heterocycles 2007, 71
(5), 1107−1115.
(57) Finnegan, W. G.; Henry, R. A.; Lofquist, R. An Improved
Synthesis of 5-Substituted Tetrazoles. J. Am. Chem. Soc. 1958, 80,
3908−3911.
(58) Charton, J.; Deprez-Poulain, R.; Hennuyer, N.; Tailleux, A.;
Staels, B.; Deprez, B. Novel non-carboxylic acid retinoids: 1,2,4-
Oxadiazol-5-one derivatives. Bioorg. Med. Chem. Lett. 2009, 19 (2),
489−492.
(59) Santilli, A.; Morris, R. L. Synthesis of 3-Arylsulfonylmethyl-1,2-
4-oxadiazole-5-carboxylic Acid Derivatives. J. Heterocycl. Chem. 1979,
16 (6), 1197−1200.
(60) Manley, P. J.; Balitza, A. E.; Bilodeau, M. T.; Coll, K. E.;
Hartman, R. C.; McFall, K. W.; Rodman, L. D.; Thomas, K. A. 2,4-
Disubstituted pyrimidines: A novel class of KDR kinase inhibitors.
Bioorg. Med. Chem. Lett. 2003, 13 (10), 1673−1677.
(61) Winkler, D.; Beconi, M.; Toledo-Sherman, L. M.; Prime, M.;
Ebneth, A.; Dominguez, C.; Munoz-Sanjuan, I. Development of LC/
̃
MS/MS, High-Throughput Enzymatic and Cellular Assays for the
Characterization of Compounds That Inhibit Kynurenine Monoox-
ygenase (KMO). J. Biol. Screening. 2013, 18 (8), 879−889.
(62) Krueger, S. K.; Williams, D. E. Mammalian flavin-containing
monooxygenases: structure/function, genetic polymorphisms and role
in drug metabolism. Pharmacol. Ther. 2005, 106, 357−387.
(63) Melo, A.; Ramos, M. J.; Floriano, W. B.; Gomes, J. A. N. F.;
Leao, J. F. R.; Magalhaes, A. L.; Maigret, B.; Nascimento, M. C.;
̃
Reuter, N. Theoretical study of arginine−carboxylate interactions. J.
Mol. Struct.: THEOCHEM 1999, 463 (1), 81−90.
Y
DOI: 10.1021/jm501350y
J. Med. Chem. XXXX, XXX, XXX−XXX