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

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.^5,6Hydrolysis 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

HD²and 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

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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⁺.⁷An 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.^8,9For example, early grade HD patients show increased

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3-HK/KYNA and QA/KYNA ratios in striatum and cortex

compared to controls.¹⁰Elevation of 3-HK levels has also been

reported in the brain of the R6/2 transgenic HD mouse

model.¹¹Other 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.¹¹Evidence 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.¹²The 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.²⁷The 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.^28,29QA can act as an NMDA receptor agonist,

with an EC₅₀of 7−200 μM for NR2A- and NR2B-containing

subunits of the NMDAR.³⁰When 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.^31,32,32,333-HK is a known reactive radical

generator³⁴and can exacerbate QA-mediated lesions.^35,36

KYNA, an antagonist of the NMDA receptor acting at the

glycine coagonist site at micromolar concentrations,³⁷has been

shown to protect against QA- and kainate-induced neuro-

toxicity.³⁸Conversely, 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 IC₅₀of 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.⁴⁰Further 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.⁴¹Removal 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.¹⁶Unlike 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.⁴²Recently this compound was found to

increase KYNA levels and be neuroprotective in a ﬂy model of

HD.¹⁵A 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.⁴³This 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,¹⁴and by pharmacological and genetic

inhibition of KMO in a ﬂy model of HD that led to increased

levels of KYNA and ameliorated neurodegeneration.¹⁵Pretreat-

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.¹⁸In the brain, KMO is mainly expressed by microglia,¹⁹

whereas KAT II is expressed predominantly in astrocytes and

neurons.²⁰Therefore, pathway metabolites such as 3-HK and

KYNA are produced in diﬀerent cells, as these branches of the

pathway are segregated.^21,22Furthermore, the interplay of the

KP between the peripheral (blood and other tissues) and

central compartments is not fully understood.²³While

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.²⁴The levels of KYNA in the brain can be inﬂuenced via

inﬂux of KYN into the brain from the periphery;²⁵for example,

systemic administration of 100 mg/kg of KYN to mice has been

shown to signiﬁcantly increase central levels of both KYN and

KYNA.²⁶But 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

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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

21.⁵⁴Palladium-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.⁵⁶Reaction 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.⁵⁷Alternatively,

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.^48,49Suzuki 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).^50,51The

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.⁶¹We

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 S_NAr 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 11.⁵³Sodium 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,⁶¹also 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.

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a

Scheme 2. Modiﬁcation via S_NAr 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 IC₅₀values 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

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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 IC₅₀of 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 IC₅₀of 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).

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Figure 5. Overview of SAR carried out on the pyrimidine scaﬀold.

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

N

0.0006

0.0032

0.09

0.033

0.17

1.1

49

50

22

23

28

27

25

24

26

H

CH

N

H

CH

N

CH₃

H

N

0.012

0.006

0.14

0.3

F

N

9.2

H

OH

N

3.5

H

OCH₃

CH₃

N

0.04

4.4

H

N

0.010

0.26

1.5

H

NH₂

N

5.5

H

CH₂OCH₃

N

0.033

4.3

a

SD = 5−10%, IC₅₀values 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,⁴³we

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%, IC₅₀values 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

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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.⁶³The 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 pK_avalues were able to maintain inhibitory activity for

KMO. The tetrazole carboxylic acid isostere can exist in two

tautomeric states; the calculated pK_avalues for these tautomeric

forms in example 31 are 3.5 and 6.9, respectively. Similarly,

oxadiazolone 35 has calculated pK_avalues of 2.93 and 8.4,

respectively. Both compounds retained KMO inhibitory activity

with IC₅₀values of 2 and 12 nM, respectively. Likewise, acidic

acyl sulfonamide 51 (calculated pK_a= 3.9) had an IC₅₀of 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,⁶⁴the 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

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a

Table 3. Replacement of the Carboxylic Acid

a

N/A = not applicable, NT = not tested, SD = 5−10%, IC₅₀values are the average of triplicate tests.

I

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a

Table 4. Structure−Activity Exploration of Phenyl Substitutions

a

NT = not tested, SD = 5−10%, IC₅₀values are the average of triplicate tests.

J

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a

Table 5. 3-Chlorophenyl Pyrimidine Acids: Optimization of the Para Position

a

NT = not tested, SD = 5−10%, IC₅₀values 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 IC₅₀of 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).⁶⁵The compounds evaluated were

not PGP substrates but were found to be substrates of BCRP.

K

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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%, IC₅₀values 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.⁵¹In 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

64

N/A

0.25

N/A

observed

_maxnorm

μM·kg/mg

1.9

0.035

C

observed

T_max

h

N/A

1.0

0.083

1.0

CL

MRT

V_dss

mL/h/kg

290

1.1

N/A

2.4

N/A

0.70

N/A

1.7

h

L/kg

h

0.32

1.8

half-life

(t_1/2

)

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 V_dssof 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 C_maxnorm 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

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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

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 T_max

half-life (t1/2)

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 EC₅₀of 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

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

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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.^24,25,66,67To evaluate

the extent of brain extracellular levels of kynurenine

metabolites, we measured compound 75 levels with quantita-

tive microdialysis using a metaquant MQ probe,⁶⁸and 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 EC₅₀for 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

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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 EC₅₀in 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

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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.²⁵In 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

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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 C₁₁H₇FN₂O₂M + 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

C₁₁H₆ClFN₂O₂M + 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 C₁₁H₆Cl₂N₂O₂M +

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 C₁₁H₇ClN₂O₂M + 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

C₁₁H₈N₂O₂M + 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 C₁₁H₆ClFN₂O₂M + 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 C₁₁H₆F₂N₂O₂M + 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 C₁₀H₇N₃O₂M + 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 C₁₁H₆F₂N₂O₂M + 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 C₁₇H₁₂N₂O₂M + 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 C₁₁H₇ClN₂O₂M + 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 C₁₂H₆ClF₃N₂O₂M + 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 C₁₁H₆Cl₂N₂O₂M + 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

C₁₂H₇F₃N₂O₂M + 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

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

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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

C₁₂H₇F₃N₂O₂M + 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 C₁₂H₈Cl₂N₂O₂M + 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 C₁₁H₅Cl₂FN₂O₂M + 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 C₁₂H₁₀N₂O₂M + 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 C₁₂H₈Cl₂N₂O₃M + 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 C₁₂H₈Cl₂N₂O₂M + 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

C₁₁H₇Cl₂N₃O₂M + 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 C₁₂H₆F₄N₂O₂M + 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

C₁₂H₉ClN₂O₂M + 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 C₁₂H₉FN₂O₂M + 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 C₁₂H₉ClN₂O₃M + 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

C₁₂H₉ClN₂O₃M + 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 C₁₄H₁₃ClN₂O₃M + 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

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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 C₁₅H₁₃ClN₂O₃M + 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;

δ_H(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

C₁₄H₁₁ClN₂O₄M + 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

C₁₂H₆ClF₃N₂O₃M + 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 C₁₆H₁₅ClN₂O₃M + 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 C₁₇H₁₇ClN₂O₃M + 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 C₁₅H₁₄ClN₃O₂M + 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

C₁₇H₁₈ClN₃O₄M + 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

C₁₇H₁₈ClN₃O₄M + 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 C₁₅H₁₃ClN₂O₃M + 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),

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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 C₁₁H₆Cl₂N₂O₃M + 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

C₁₁H₁₄N₂O₂M + 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 C₁₁H₅Cl₂N₃M + 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 C₉H₁₁N₃O₃M + 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 C₁₁H₇Cl₂N₃O₂M + 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

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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 C₁₂H₆Cl₂N₄O₂M + 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 C₁₁H₆Cl₂N₆M + 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 C₁₂H₈Cl₂N₆M + 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 C₁₃H₈Cl₂N₄M + 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,

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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 C₁₃H₈Cl₂N₄M + 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 C₁₂H₇Cl₂NO₂M + 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 C₁₂H₇Cl₂N₂O₂M + 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. t_R= 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. t_R= 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. t_R= 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

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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. t_R= 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 C₁₃H₇ClN₂O₃M + 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

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ASSOCIATED CONTENT

* Supporting Information

■

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