Optimization of a Novel Quinazolinone-Based Series of Transient Receptor Potential A1 (TRPA1) Antagonists Demonstrating Potent in Vivo Activity

Article

pubs.acs.org/jmc

Optimization of a Novel Quinazolinone-Based Series of Transient

Receptor Potential A1 (TRPA1) Antagonists Demonstrating Potent in

Vivo Activity

Laurie B. Schenkel,*^,†Philip R. Olivieri,^†Alessandro A. Boezio,^†Holly L. Deak,^†Renee Emkey,^‡

Russell F. Graceﬀa,^†Hakan Gunaydin,^§Angel Guzman-Perez,^†Josie H. Lee,^‡Yohannes Teﬀera,^∥

Weiya Wang,^⊥Beth D. Youngblood,^⊥Violeta L. Yu,^‡Maosheng Zhang,^⊥Narender R. Gavva,^⊥

Sonya G. Lehto,^⊥and Stephanie Geuns-Meyer^†

^†Departments of Medicinal Chemistry, ^‡Lead Discovery, ^§Molecular Structure, and ^∥Pharmacokinetics and Drug Metabolism, Amgen,

Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States

^⊥Neuroscience, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States

S

* Supporting Information

ABSTRACT: There has been signiﬁcant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the

treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identiﬁcation of a potent small

molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we

describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening

identiﬁed 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing

polarity in order to improve intrinisic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent,

selective antagonist of TRPA1 with pharmacokinetic properties allowing for >30-fold coverage of the rat TRPA1 IC₅₀in vivo.

Compound 27 demonstrated dose-dependent inhibition of AITC-induced ﬂinching in rats, validating its utility as a tool for

interrogating the role of TRPA1 in in vivo pain models.

A number of preclinical studies provide additional support

for the role of TRPA1 in pain. TRPA1 knockout mice

demonstrate signiﬁcantly diminished ﬂinching behavior upon

exposure to the selective agonist AITC,²as well as to formalin⁷

and the inﬂammatory mediator 4-hydroxynonenal.⁸Knockout

mice also respond less to the application of tactile force.⁶

Pharmacological blockade of TRPA1 with antagonists, such as

xanthine HC-030031 (1)⁹and oxime AP-18 (2),¹⁰have shown

eﬃcacy in formalin- and complete Freund’s adjuvant (CFA)-

induced models of inﬂammatory pain and the spinal nerve

ligation (SNL) model of neuropathic pain (Figure 1). While

these results demonstrate the potential utility of treating pain

with small molecule TRPA1 antagonists, the weak potencies

(3−10 μM) and/or poor pharmacokinetic properties of these

INTRODUCTION

■

TRPA1 is a nonselective cation channel and member of the

transient receptor potential (TRP) superfamily of ion channels.

TRPA1 is highly expressed in sensory neurons of the dorsal

root ganglia and is activated by a range of irritants. Reactive

electrophiles such as allyl isothiocyanate (AITC), allicin, and

cinnamaldehyde, the pungent components of mustard oil,

garlic, and cinnamon, respectively, activate the channel via

covalent modiﬁcation of cysteine residues and elicit a painful

response in humans.¹Sensory neurons from TRPA1-deﬁcient

mice show diminished response to these types of irritants,

which strongly supports the role of TRPA1 in mediating their

pain response.²TRPA1 can also be activated via noncovalent

mechanisms, including exposure to unreactive small molecules

such as gingerol³and thymol,⁴inﬂammatory peptides,⁵cold

temperature,²and mechanical force.⁶

Received: January 8, 2016

A

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Figure 1. Previously reported TRPA1 antagonists and proﬁle of HTS hit 4.

a

Table 1. SAR of the Quinazolinone Amides

b

c

compd

R

R¹

rTRPA1 IC₅₀(μM)

hTRPA1 IC₅₀(μM)

RLM/HLM Cl_{int, app}(μL/min/mg)

cLogP

4

8-Me

H

p-OCHF₂

H

0.142

0.685

>399/194

ND/ND

706/224

104/31

2.9

2.6

2.5

3.3

2.8

3.3

3.7

2.9

3.0

2.3

1.7

de

,

e

5

U

>8.33

e

de

,

6

p-OMe

m-OMe

o-OMe

p-Cl

1.37

U

e

7

>16.7

0.358

>16.7

0.802

>16.7

8

9

de

,

e

10

11

12

13

14

15

16

17

18

19

p-Cl

U

e

7-Me

6-Me

5-Me

8-CF₃

8-F

p-Cl

>16.7

0.287

0.112

>4.2

829/144

ND/ND

736/301

>399/397

186/40

p-Cl

0.265

0.070

p-Cl

e

p-Cl

0.306

0.791

0.286

e

p-Cl

1.33

e

8-OMe

8-CN

8-aza

7-aza

p-Cl

2.15

1.46

455/34

p-Cl

0.056

1.17

1.08

0.201

1.18

1.71

297/49

p-Cl

18/<14

p-Cl

179/14

a

Data represent the mean of ≥2 independent dose−response curves, except where noted. Standard deviation values are provided in the Supporting

b

c

d

Information. Antagonist IC₅₀, FLIPR assay. ND = not determined. U = undeﬁned; inhibition crossed the 50% threshold, but a curve could not be

ﬁt. Data represents n = 1 dose−response curve.

e

and other ﬁrst generation antagonists make it challenging to

assess whether the observed in vivo eﬀects are truly mediated

by TRPA1, as signiﬁcant target coverage was diﬃcult to

achieve. A recently reported (triﬂuoromethylphenyl)indazole

antagonist (3) showed good potency (<0.1 μM) against

TRPA1 across species and good selectivity over other TRP

channels (Figure 1).¹¹Importantly, dose-dependent reversal of

both CFA-induced mechanical hyperalgesia in rats and AITC-

induced allodynia in mice was demonstrated upon oral dosing.

However, broader selectivity against a range of oﬀ-targets and

in vivo free drug concentrations were not reported.

The discovery of a human genetic link between TRPA1 and

familial episodic pain syndrome provides still further evidence

that TRPA1 plays a signiﬁcant role in human pain.¹²Patients

carrying a single gain-of-function mutation in TRPA1

experience debilitating bouts of upper body pain, triggered by

some combination of fasting, cold, and fatigue. This data,

combined with the numerous studies performed in rodents by

either genetic or pharmacological knockdown of TRPA1, and

the need for a potent, selective TRPA1 antagonist with

pharmacokinetics enabling signiﬁcant target coverage in vivo,

prompted us to initiate a program to identify such a compound.

Here we report the discovery of a quinazolinone-based series of

TRPA1 antagonists, optimization of which led to the

identiﬁcation of a compound possessing all of the features

desired to evaluate the in vivo pharmacology of TRPA1.

RESULTS AND DISCUSSION

■

To validate the hypothesis that inhibition of TRPA1 is a

suitable approach for the treatment of pain, we sought to

identify a potent and selective tool compound that could

provide good target coverage in vivo with oral dosing. High-

throughput screening identiﬁed compound 4, which possessed

submicromolar potency against both human and rat TRPA1 in

a FLIPR Ca²⁺imaging assay that measures inhibition upon

activation with AITC (Figure 1). Compound 4 showed only

antagonist activity and was inactive in agonist mode at

concentrations up to 25 μM. Despite its poor microsomal

stability, 4 was an attractive hit based on its potency, selectivity

against a small subset of other TRP channels, good physical

properties, and a modular synthesis that would enable rapid

analogue preparation and determination of SAR.

B

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Figure 2. (A) Predicted relative interaction energies for a theoretical π-stacking interaction and measured human TRPA1 potencies. (B) Plot

showing the correlation of the values in (A). Each point corresponds with a compound. Compound 10 is excluded.

Figure 3. Plot demonstrating improvements in LipE for a subset of key compounds.

We ﬁrst determined initial SAR around the phenyl ring

(Table 1). Removal of the diﬂuoromethoxy group was not well

tolerated, as compound 5 lost signiﬁcant activity. Seeking to

ﬁnd a potent, more readily available replacement for the

diﬂuoromethoxy moiety, we prepared compound 6. However,

replacement with a methoxy group also led to a loss of potency.

Para-substitution was important for maintaining detectable

activity in both species, as both 7 and 8 showed no inhibition at

concentrations up to 16.7 μM. A scan of small substituents at

the para-position identiﬁed 9, which largely restored the

potency against both rat and human TRPA1 observed for 4,

thus the p-Cl-phenyl moiety was selected for further

optimization.

well-tolerated at all but the 7-position of the quinazolinone.

Despite the promising potency of these compounds, their

microsomal stability remained poor. The relatively good

microsomal stability of 10 compared to the methyl-substituted

compounds suggested that the methyl groups were likely to be

metabolic soft spots. Replacement of the 8-methyl substituent

with the triﬂuoromethyl group in 14 provided a signiﬁcant

improvement with respect to potency but with similarly poor

metabolic stability. The ﬂuorinated compound 15 was not as

potent as 14 but was signiﬁcantly more intrinsically stable in

both rat and human liver microsomes.

In addition to methyl, triﬂuoromethyl, and ﬂuoro, the

methoxy substituent in 16 also provided a relatively potent

inhibitor. On the basis of the apparent potency enhancement

provided by either electron-withdrawing or election-donating

substituents, we hypothesized that the quinazolinone core may

engage TRPA1 via a π-stacking interaction. Published

computational models of the interaction energies of benzene

A set of compounds was next prepared to examine SAR

around the phenyl ring of the quinazolinone core. The poor

activity of 10 indicated that the methyl substitution on the

phenyl ring was important for the potency of 9, and data for

11−13 indicated that substitution with a methyl group was

C

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c

Table 2. SAR of Oxadiazole Analogues

a

b

c

Antagonist IC₅₀, FLIPR assay. Data represents n = 1 dose−response curve. Data represent the mean of ≥2 independent dose−response curves,

except where noted. Standard deviation values are provided in the Supporting Information.

dimers suggest that any substituent enhances π-stacking

interaction energy relative to the unsubstituted benzene

dimer.¹³Assuming that the model system predicts the

interaction between the substituted benzene ring of an inhibitor

and an aromatic residue of the target protein, the relative

enhancement of various substituents can be predicted. Indeed,

while the precise potency gains over the unsubstituted parent

10 cannot be determined due to its lack of measurable activity,

the potencies observed for 9, 14, 15, and 16 align well relative

to each other when compared with the predicted values (Figure

2).¹⁴

Increasing polarity is a common strategy for improving the

microsomal stability of compounds that are oxidatively

metabolized; thus, we sought to decrease the cLogP of the

quinazolinones. This strategy was well aligned with further

testing of the π-stacking interaction hypothesis, as both nitrile

and pyridine substitutions are predicted to have a favorable

impact on the π-stacking contribution to potency and would

signiﬁcantly increase polarity. We were pleased to see that

nitrile 17 was more potent than earlier compounds, and its

cLogP was signiﬁcantly lower than that of 9−16 (Table 1). As

anticipated, the stability of 17 was also improved in rat liver

microsomes versus most previously prepared compounds and

the relative stability in human liver microsomes observed for 15

and 16 was maintained. Encouraged by this result, we sought to

further reduce cLogP by preparing azaquinazolinones 18 and

19. Though 18 suﬀered a decrease in potency relative to 17, its

microsomal stability was promising, as this was the ﬁrst example

of a compound showing very low turnover in both human and

rat microsomes. The increased polarity of 18 alone does not

account for its improved intrinsic stability. Compound 19,

which has an identical cLogP, was signiﬁcantly less stable in rat

liver microsomes. This suggests that the position of the

nitrogen atom in 18 may be key for reducing oxidative

metabolism. Notably, the potency data for the nitrile- and

pyridine-containing analogues further supports the hypothesis

that the core engages TRPA1 through a π-stacking interaction

(Figure 2). Though 18 was less potent than HTS hit 4, its

lipophilic eﬃciency (LipE = 4.3) was improved versus 4

(Figure 3). The azaquinazolinone core was retained for further

optimization due to its beneﬁcial eﬀect on microsomal stability,

which had been challenging to improve in the parent

quinazolinone series.

Having completed initial SAR at both ends of the scaﬀold, we

next turned our attention toward optimization of the amide

moiety. A small series of analogues (20−22) was prepared that

D

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c

Table 3. SAR of Analogues Modiﬁed To Reduce Metabolism at the Benzylic Position

a

b

c

Antagonist IC₅₀, FLIPR assay. Data represents n = 1 dose−response curve. Data represent the mean of ≥2 independent dose−response curves,

except where noted. Standard deviation values are provided in the Supporting Information.

incorporated oxadiazole isomers as replacements for the amide

(Table 2). We were pleased to ﬁnd that the oxadiazoles in 20

and 21 restored or exceeded the potency against both rat and

human TRPA1 that had been observed for the most potent

quinazolinone amides, with only a relatively small loss of

microsomal stability. With cLogP values that are roughly

equivalent to amide 18, the improved potency of compounds

20 and 21 provided signiﬁcant increases in LipE (5.5 and 6.1,

respectively) over 18 (Figure 3). Oxadiazole isomer 22 was not

well tolerated. Because the oxadiazole isomer in 21 was

approximately 4-fold more potent than isomer 20, it was

selected for further optimization.

Analogues 23−25 were prepared, all of which contain an

additional nitrogen atom in the core. None of these compounds

showed an improvement in TRPA1 potency and, despite their

equivalent increased polarity, only 25 showed an improvement

in microsomal stability. A methyl group was also incorporated

at the 5-position of the azaquinazoline core to give compound

26, as substitution at this position had previously provided a

signiﬁcant improvement in potency. While a moderate potency

improvement was observed in human TRPA1 for 26 versus 21,

a signiﬁcant deterioration in microsomal stability was also

observed, which may be expected due to its increase in cLogP

and the reintroduction of a likely metabolic soft spot. Similar

results were observed on both potency and stability when a

methyl group was incorporated at the 2-position of the

azaquinazolinone core.¹⁶Seeking a diﬀerent approach to

increase polarity, compound 27 (AM-0902) was prepared,

which has a low cLogP and also places a methyl group in the

space that had repeatedly proven beneﬁcial for TRPA1 activity.

Indeed 27 showed good potency against both TRPA1 isoforms,

thereby demonstrating a signiﬁcant improvement in LipE (6.8)

versus earlier analogues (Figure 3), and had very good

microsomal stability. The observed improvement in potency

also supports the π-stacking interaction hypothesis put forward

for earlier compounds, as imidazole is predicted to signiﬁcantly

improve a theoretical π-stacking interaction relative to

substituted phenyl rings or pyridine (Figure 2).¹⁴Compound

28, which contains the alternate oxadiazole isomer, showed a

similar decrease in potency that was observed previously

between 20 and 21. The pyrazole isomer 29 had signiﬁcantly

less microsomal stability with similar cLogP, suggesting, in

analogy to compounds 18 and 19, that the speciﬁc arrangement

of nitrogen atoms in 27 may be critical for reducing oxidative

metabolism.

We reasoned that incorporating additional polarity into the

phenyl ring might further improve microsomal stability, but

polarity was not well tolerated in this region. This trend is

exempliﬁed by compound 30 (Table 3). A signiﬁcant decrease

in potency was observed when compared with phenyl analogue

21, though as anticipated microsomal stability did improve

substantially.

Metabolite identiﬁcation studies in rat and human liver

microsomes with 20 and 21 determined that the benzylic

E

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Table 4. Potency of Selected Analogues in the ⁴⁵Ca²⁺Flux

Assay

position was the major site of oxidative metabolism. Alcohol 31,

identiﬁed as the major metabolite of 21 in microsomes,

retained good potency against both rat and human TRPA1 and

showed signiﬁcantly improved microsomal stability (Table 3).

Other compounds in which the benzylic position was blocked

with less polar substituents, as in 32 and 33, were relatively

potent inhibitors, but the metabolic stability was either

comparable to the parent compound (32 versus 21) or

substantially worse (33 versus 20).

Hypothesizing that an oleﬁn may mimic the binding

conformation of the saturated analogues while also mitigating

oxidative metabolism at the benzylic position, we calculated the

lowest energy conformations of the trans-disubstituted and

trisubstituted oleﬁns.¹⁵An overlay with the low energy

conformation of the saturated analogue 20 (Figure 4)

a

rTRPA1 IC₅₀

hTRPA1 IC₅₀rTRPA1 EC₅₀hTRPA1 EC₅₀

b c c

b

compd

(μM)

4

2.62

6.27

1.58

>47

>40

>47

>40

20

21

27

31

35

37

0.411

0.120

0.071

0.204

0.053

0.055

0.376

0.131

0.251

0.085

0.063

a

Data represent the mean of ≥2 independent dose−response curves.

b

c

Antagonist IC₅₀, ⁴⁵Ca²⁺ﬂux assay. Agonist EC₅₀, ⁴⁵Ca²⁺ﬂux assay.

oxidation, 20 also demonstrated moderate oral bioavailability.

Despite these improvements, at C_max20 achieved only 3-fold

unbound coverage over its ⁴⁵Ca²⁺IC₅₀following a 30 mpk oral

dose. Compound 27, while cleared in an iv PK experiment at

nearly the same rate as 20, showed a signiﬁcantly lower

unbound clearance (CL/fu) due to its larger free fraction.

While total exposure and oral bioavailability for 20 and 27 were

similar, 27 had a higher unbound C_maxand signiﬁcantly better

potency, which enabled achievement of unbound concen-

trations that were 31-fold over the ⁴⁵Ca²⁺IC₅₀following a 30

mg/kg oral dose. Compound 27 is highly permeable (average

Figure 4. Overlay of the calculated low energy conformations of 20

(gray), 34 (turquoise), and 35 (orange).

suggested that an oleﬁn, and in particular the trisubstituted

oleﬁn, may position the critical p-chlorophenyl substitutent in a

similar orientation as the saturated compounds. Indeed, while

the potency of 34 was signiﬁcantly decreased relative to 20, the

trisubstituted oleﬁn 35 restored the potency of the parent

compound 20 and improved microsomal stability substantially.

The trisubstituted oleﬁn was also well tolerated in the

imidazopyrimidinone core, as exempliﬁed by 36 and 37.

Compound 37 was especially noteworthy, as it exhibited sub-

100 nM potency against both the rat and human TRPA1

channels with less than detectable turnover in both rat and

human liver microsomes. While oleﬁns are typically prone to

oxidative metabolism, it is likely that the observed microsomal

stability of 34−37 is due to the combination of steric hindrance

and electron deﬁciency present in these oleﬁn analogues.

The potencies of compounds of interest were also

determined in a radioactive calcium ﬂux assay, which is

considered to be a direct measure of TRPA1 potency, as it

quantiﬁes only inhibition of ⁴⁵Ca²⁺entering the cell upon

activation and opening of the channel with AITC. In contrast,

the FLIPR assay measures changes in cellular Ca²⁺levels that

can arise from both extracellular and intracellular sources. Data

for compounds 4, 20, 21, 27, 31, 35, and 37 show that, despite

a right-shift for several of these compounds, the potency

observed in the FLIPR assay was generally well maintained in

the ⁴⁵Ca²⁺ﬂux assay (Table 4).

P

_app= 44.5 μcm/s in MDCK cells), an unlikely substrate for P-

gp (eﬄux ratio = 1.3 in P-gp overexpressing MDCK cells), and

demonstrates good solubility (PBS pH 7.4: 226 μM, SIF: 248

μM).

The oleﬁns 35 and 37 showed signiﬁcantly diﬀerent PK

proﬁles than their saturated analogues. Though low clearance

was observed for 35 following iv dosing, its very small free

fraction provided an unbound clearance comparable to that of

20. Consequently, neither 35 nor 37 achieved high multiples

over the ⁴⁵Ca²⁺IC₅₀following oral dosing, despite their total

exposures being much higher and half-lives much longer than

that of the saturated analogues.

Additional proﬁling of compound 27 was performed to

assess its selectivity. Compound 27 showed good selectivity

over other TRP channels, as no activity was observed against

human TRPV1 or TRPV4, or rat TRPV1, TRPV3, or TRPM8,

at concentrations up to 10 μM. Activity of 27 was also assessed

in a CEREP panel of 40 targets at 10 μM concentration to

identify oﬀ-target activities, and little to no inhibition was

observed (>77 POC against all targets).¹⁶No activity was

detected in CYP 3A4 or CYP2D6 assays (est IC₅₀> 27 μM for

both). Importantly 27 showed only 10% block of Na_v1.7

channels at 10 μM in mouse DRG neurons, which suggested

that any potential eﬃcacy observed in an in vivo pain model

would not be due to inhibition of Na_v1.7.¹⁷

Protein binding and rat PK data for a subset of analogues are

summarized in Table 5. Anticipating that a tool compound used

to interrogate the role of TRPA1 in pain would be dosed in

acute pain models, we sought to identify a compound that

would provide at least 10-fold unbound coverage over its ⁴⁵Ca²⁺

IC₅₀at maximum concentration (C_max) following a moderate

dose. A single-dose intravenous (iv) PK experiment showed

that compound 21 was cleared at a rate exceeding rat liver

blood ﬂow. While also relatively high, compound 20 showed

improved iv clearance relative to 21. As expected for a

compound that is both well absorbed and cleared via hepatic

We also measured inhibition of TRPA1 activity induced by

agents other than AITC. Compound 27 inhibited ⁴⁵Ca²⁺ﬂux

upon activation of rat TRPA1 with methylglyoxal, an

endogenous and selective agonist,¹⁸with an IC₅₀of 0.019

μM. Inhibition of ⁴⁵Ca²⁺ﬂux was also measured upon activation

of rat TRPA1 by changes in osmolarity, which may mimic

mechanical activation of the channel,¹⁹and 27 returned an IC₅₀

of 0.010 μM. The potency of compound 27 in these assays

showed that its antagonism of TRPA1 was not conﬁned solely

to AITC activation and suggested that its activity was likely to

extend into physiologically relevant contexts.

F

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Table 5. Protein Binding and Pharmacokinetic Data in Rat for Selected Analogues Following Intravenous and Oral

Administration

a

b

iv

oral

compd rat ppb (Fu) CL (L/h/kg)

CL_uVss (L/kg) T_1/2(h) AUC_inf(μM-h) C_max(C_maxu) (μM) T_max(h) T_1/2(h)

F%

C_maxu/⁴⁵Ca²⁺IC₅₀

20

21

27

35

37

0.093

0.144

0.289

0.012

0.033

2.2

24

NA

9

1.0

2.0

1.7

1.4

ND

0.4

0.3

0.6

4.2

ND

19.2

ND

10.3 (1.0)

ND

0.5

ND

0.8

2.7

2.0

4.0

52

3

c

5.9

ND

2.8

ND

60

−

31

2

4

c

2.5

19.8

94.9

117.0

7.5 (2.2)

8.0 (0.1)

6.5 (0.2)

0.27

ND

b

22

−

5.1

32

10.0

ND

a

0.5 mg/kg, 100% DMSO. 30 mg/kg for all except 37 (10 mg/kg), 1% Tween 80/2% HPMC/97% water/methanesulfonic acid pH 2.2. Cl_unot

calculated, as clearance of high extraction ratio compounds is primarily blood-ﬂow dependent.

Figure 5. Summary of AITC ﬂinching in rats upon oral administration of 27. (A) Dose-dependent reduction in ﬂinching. (B) Exposure−response

relationship. Each point represents an individual animal.

a

Scheme 1. Synthesis of Amides

a

39b and 39k were purchased from commercial suppliers. Reagents and conditions: (a) formamide, μW, 175 °C, 1 h; (b) Pd(PPh₃)₄, Zn(CN)₂,

DMF, 100 °C; (c) methyl 2-bromoacetate, K CO , DMF, 50 °C; (d) LiOH, THF/H O, RT; (e) T3P, Hunig’s base, EtOAc, RT; (f) 2-bromo-N-(4-

̈

2

3

2

chlorophenethyl)acetamide (40), K₂CO₃, DMF, 50 °C.

The combination of its potent TRPA1 inhibition, oral PK

enabling an acceptable projected target coverage, and excellent

selectivity suggested that compound 27 would be a suitable tool

compound to assess the pharmacology of TRPA1 in vivo. To

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a

Scheme 2. Synthesis of Oxadiazoles 20, 28, and 33−36

a

Reagents and conditions: (a) bromoacetonitrile, K₂CO₃, DMF, 50 °C, 3 h; (b) hydroxylamine, EtOH, 80 °C, 2 h; (c) carboxylic acid 44, 45, 46, or

47, T3P, Et₃N, EtOAc, 80 °C, 3 h.

a

Scheme 3. Synthesis of Oxadiazoles 21, 23−26, and 29

a

Reagents and conditions: (a) hydroxylamine, EtOH, 80 °C, 2 h; (b) N-Boc-glycine, Et₃N, T3P, EtOAc, 80 °C, 5 h; (c) TFA, DCM, RT, 17 h; (d)

HOBt, EDC·HCl, heteroarylamino acids, Hunig’s Base, DMF, RT, 4 h, then CH(OMe) , AcOH, 100 °C, 17 h.

̈

3

a

Scheme 4. Synthesis of Oxadiazoles 27, 30−32, and 37

a

Reagents and conditions: (a) hydroxylamine, EtOH, 80 °C, 2 h; (b) tert-butyl bromoacetate, K₂CO₃, DMF, 50 °C, 6 h; (c) concentrated HCl, RT,

30 min; (d) 50, CDI, DMF, 50−100 °C, 1 h; (e) DAST, DCM, 0 °C to RT, 1 h.

that end, compound 27 was advanced into an in vivo model

using AITC-induced ﬂinching in rats (Figure 5), which has

been reported²⁰as a useful method for measuring TRPA1

target engagement. Rats were dosed orally with either vehicle

(2% HPMC/1% Tween-80) or the TRPA1 antagonist 27 at 1,

3, 10, or 30 mg/kg. After 1 h, one left ventral hind paw was

injected with the TRPA1 agonist AITC (0.1%). Nocifensive

behavior was then observed and recorded during the ﬁrst

minute postinjection. A dose-dependent reduction of AITC-

induced ﬂinching was observed for 27, with a signiﬁcant

reduction in ﬂinching observed postdosing of 10 and 30 mg/kg.

The unbound plasma concentrations (C_u) at 1 h for the 1, 3,

10, and 30 mg/kg doses were 0.051 0.024 (n = 8), 0.19

0.11 (n = 8), 0.58 0.35 (n = 8), and 2.2 0.40 (n = 8) μM,

covering the in vitro rat TRPA1 ⁴⁵Ca²⁺IC₅₀at 0.72, 2.7, 8.2,

and 30.3 fold, respectively. A good exposure−response

relationship was observed in this target coverage model (Figure

5). An unbound in vivo IC₅₀of 0.35 μM, which is in good

agreement with the in vitro rat TRPA1 ⁴⁵Ca²⁺IC₅₀, and

unbound in vivo IC₉₀of 1.7 μM were determined. It is

noteworthy that at a dose of 30 mg/kg, 27 engages TRPA1 at

concentrations that exceed the in vivo IC₉₀, making it a useful

tool for exploration of in vivo models of acute pain.

CHEMISTRY

■

Final compounds containing amides were prepared according

to the synthesis shown in Scheme 1. Commercially available

acids 38 were converted to the corresponding quinazolinone

intermediates 39a, 39c−h, and 39j via condensation with

formamide. 39i was prepared via cyanation of the bromide

intermediate. Intermediates 39a,b and 39f,g were converted to

carboxylic acids 41a−d by regioselective alkylation with methyl

2-bromoacetate followed by saponiﬁcation with LiOH. T3P-

mediated amide coupling then provided ﬁnal compounds 5−

10, 14, and 15. Regioselective alkylation of 39c−e and 39h−k

with 2-bromo-N-(4-chlorophenethyl)acetamide 40 provided

compounds 11−13 and 16−19.

Oxadiazoles 20, 28, and 33−37 were prepared according to

Scheme 2. Intermediates 39j and 39l¹⁶were converted to

nitriles 42a,b by regioselective alkylation with bromoacetoni-

trile. Addition of hydroxylamine to the nitriles provided

intermediates 43a,b, which were converted to the oxadiazoles

20, 28, and 33−37 via T3P-mediated condensation with

carboxylic acids 44−47.

The oxadiazoles 21, 23−26, and 29 were prepared according

to Scheme 3. Addition of hydroxylamine to 3-(4-chlorophenyl)-

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coupling constants (in Hz), and number of protons. Purity for all ﬁnal

compounds was >95% and was measured using one of two Agilent

LCMS systems. The ﬁrst used a HALO C18 from Advanced Materials

Technology, 3.0 × 50 mm, 2.7 μm column, gradient: 5−95% B over

1.5 min, 1.9 min total run time, 2 mL/min, solvent A: water w/0.1%

TFA, solvent B: acetonitrile w/0.1% TFA. The second used a

CORTECS C18+ from Waters, 3.0 × 50 mm, 2.7 μm column,

gradient: 5−95% B over 1.5 min, 2.0 min total run time, 2 mL/min,

solvent A: water w/0.1% formic acid, solvent B: acetonitrile w/0.1%

formic acid. All ﬁnal compounds were ≥95% purity.

propanenitrile (49a), followed by T3P-mediated condensation

with N-Boc-glycine and deprotection with TFA, provided the

key intermediate 48. Condensation of 48 with commercially

available heteroarylamino acids gave the intermediate amides,

which were then condensed with trimethyl orthoformate in

acetic acid to provide the ﬁnal compounds.

The oxadiazoles 27, 30−32, and 37 were prepared using the

alternate route detailed in Scheme 4. Intermediates 39j and 39l

were converted to the carboxylic acids 41e and 41f via

regioselective alkylation with tert-butyl bromoacetate followed

by removal of the tert-butyl group under acidic conditions. The

nitrile intermediates 49a−d were treated with hydroxylamine to

provide 50a−d. CDI-mediated condensation of 50a−d with

either 41d or 41e provided ﬁnal compounds 27, 30, 31, and 37.

Compound 31 was converted to 32 by treatment with DAST.

Oxadiazole 22 was prepared by T3P-mediated condensation

of intermediate 41d with 51 (Scheme 5).

All in vivo procedures described in this manuscript were approved

by the Institutional Animal Care and Use Committee at Amgen

(Thousand Oaks, CA).

General Procedure A: N-(4-Chlorophenethyl)-2-(8-methyl-4-ox-

oquinazolin-3(4H)-yl)acetamide (9). A vial was charged with 2-(8-

methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (41a) (75 mg, 0.344

mmol), 2-(4-chlorophenyl)ethanamine (0.064 mL, 0.412 mmol),

Hunig’s base (0.150 mL, 0.859 mmol), EtOAc (0.678 mL), and 1-

̈

propanephosphonic acid cyclic anhydride, 50 wt % solution in EtOAc

(0.656 mL, 1.031 mmol), and the mixture was stirred at RT. The

mixture became homogeneous initially and then solids crashed out.

After 2 h, the solids were ﬁltered and washed with EtOAc to provide

a

Scheme 5. Synthesis of Oxadiazole 22

1

the desired compound (43 mg, 35% yield) as an oﬀ-white solid. H

NMR (400 MHz, DMSO-d₆) δ 8.35 (t, J = 5.53 Hz, 1 H) 8.28 (s, 1 H)

7.95−8.03 (m, 1 H) 2.55 (s, 3 H) 7.67−7.73 (m, 1 H) 7.43 (t, J = 7.63

Hz, 1 H) 7.31−7.37 (m, 2 H) 7.22−7.28 (m, 2 H) 4.54−4.67 (m, 2

H) 3.27−3.34 (m, 2 H) 2.73 (t, J = 7.14 Hz, 2 H). MS, m/z 356.0 [M

+ H]⁺.

a

Reagents and conditions: (a) T3P, Et₃N, EtOAc, 90 °C, 17 h.

General Procedure B: N-(4-Chlorophenethyl)-2-(5-methyl-4-

oxopyrido[2,3-d]pyrimidin-3(4H)-yl)acetamide (13). In a round-

bottom ﬂask 5-methylquinazolin-4(3H)-one (39e) (500 mg, 3.12

mmol) was dissolved in 6 mL of DMF. 2-Bromo-N-(4-

chlorophenethyl)acetamide (40) (1.02 g, 3.74 mmol) and potassium

carbonate (646 mg, 4.68 mmol) were added, and the mixture was

stirred at 50 °C for 2 h. The mixture was poured into water. The solids

were ﬁltered, washed with pentane, and dried over sodium sulfate to

provide the title compound as an oﬀ-white solid (940 mg, 85% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (t, J = 5.4 Hz, 1 H) 8.22 (s, 1

CONCLUSIONS

■

Optimization of high-throughput screening hit 4 led to

identiﬁcation of a potent series of purinone oxadiazole

TRPA1 inhibitors. A key strategy for hit optimization was to

minimize microsomal turnover by increasing polarity, thereby

improving LipE. Design strategy was also informed by a π-

stacking hypothesis that may account for the observed potency

SAR around the quinazolinone core, and by modeling of low

energy conformations, which enabled discovery of the potent

and stable trisubstituted oleﬁn analogues. Compound 27 (AM-

0902), which is a potent TRPA1 inhibitor across multiple

modes of channel activation, is a selective antagonist with

pharmacokinetic properties conducive to oral dosing in in vivo

target validation studies. Compound 27 showed dose-depend-

ent and nearly complete reduction of AITC-ﬂinching in rats,

thereby demonstrating excellent in vivo target coverage. On the

basis of published data, 27 may be superior to other known tool

compounds reported to date, and disclosure of the eﬃcacy of

27 in in vivo pain models is forthcoming.

H) 7.67 (t, J = 7.77 Hz, 1 H) 7.50 (d, J = 8.0 Hz, 1 H) 7.34 (t, J = 7.8

Hz, 2 H) 7.28 (t, J = 7.6 Hz, 2 H) 4.55 (s, 2 H) 3.30 (m, 3 H) 2.77 (s,

3 H) 2.73 (d, J = 7.1 Hz, 2 H). MS, m/z 356.1 [M + H]⁺.

General Procedure C: 3-((5-(4-Chlorophenethyl)-1,2,4-oxadiazol-

3-yl)methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (20). A vial was

charged with 3-(4-chlorophenyl)propionic acid (44) (60.6 mg, 0.328

mmol,), (Z)-N′-hydroxy-2-(4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)-

acetimidamide (43a) (72 mg, 0.328 mmol), ethyl acetate (1.31 mL),

and triethylamine (0.183 mL, 1.314 mmol). 1-propanephosphonic acid

cyclic anhydride, 50 wt % solution in ethyl acetate (0.836 mL, 1.314

mmol) was added, and the reaction was heated at 80 °C for 3 h. Water

and additional EtOAc were added, and the layers were separated. The

organic portion was dried over sodium sulfate, ﬁltered, and

concentrated in vacuo. The crude material was puriﬁed by silica gel

chromatography with a gradient of 0−50% 90/10 DCM/MeOH in

DCM to provide the title compound (67 mg, 0.182 mmol, 55.5%

EXPERIMENTAL SECTION

■

1

Chemistry. Unless otherwise noted, all materials were obtained

from commercial suppliers and used without further puriﬁcation.

Compound 4 was purchased from Enamine, and compounds 39b, 39k,

44, 46, and 49a were purchased from common suppliers. Anhydrous

solvents were obtained from Aldrich or EM Science and used directly.

All microwave-assisted reactions were conducted with Biotage Initiator

systems. Silica gel chromatography was performed using prepacked

silica gel cartridges and Biotage Isolera One normal phase

chromatography systems. Reverse phase chromatography was

performed on either a Biotage Isolera Four or Gilson GX-281 system.

¹H NMR spectra were recorded on either a Bruker AVANCE-400 (at

400.13 MHz) or a Bruker AVANCE III-500 (at 500.34 MHz)

spectrometer at ambient temperature. Chemical shifts are reported in

parts per million (ppm, δ units) relative to residual proton signal in the

solvent. Data are reported as follows: chemical shift, mulitplicity (s =

singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet),

yield) as a white solid. H NMR (400 MHz, DMSO-d₆) δ ppm 9.02

(dd, J = 4.60, 2.05 Hz, 1 H), 8.55 (dd, J = 7.92, 1.86 Hz, 1 H) 8.74 (s,

1 H), 7.62 (dd, J = 7.92, 4.60 Hz, 1 H), 7.25 (d, J = 4.01 Hz, 4 H), 5.37

(s, 2 H), 3.21−3.27 (m, 2 H), 2.98−3.05 (m, 2 H). MS, m/z 367.9 [M

+ H]⁺.

General Procedure D: 3-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-

5-yl)methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (21). A vial was

charged with 2-aminonicotinic acid (87 mg, 0.631 mmol), (3-(4-

chlorophenethyl)-1,2,4-oxadiazol-5-yl)methanamine (48) (150 mg,

0.631 mmol), HOBt (145 mg, 0.947 mmol), DMF (2524 μL), and

Hunig’s base (441 μL, 2.52 mmol). EDC·HCl (181 mg, 0.947 mmol)

̈

was added, and the reaction was stirred at RT for 4 h. Water and

EtOAc were added, and the layers were separated. The organic portion

was dried over sodium sulfate, ﬁltered, and concentrated. The crude

material was slurried in trimethyl orthoformate (1390 μL, 12.58

mmol) and acetic acid (72.0 μL, 1.258 mmol), and the mixture was

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heated at 100 °C for 17 h. Water and EtOAc were added, and the

layers were separated. The organic portion was dried over sodium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed by

silica gel chromatography, using a gradient of 0−50% 90/10 DCM/

MeOH in DCM. The product containing fractions were combined and

concentrated. The solids were triturated in diethyl ether, ﬁltered, and

air-dried to provide the title compound as an oﬀ-white solid (163 mg,

0.443 mmol, 71% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 9.03 (dd,

J = 4.60, 2.05 Hz, 1 H) 8.74−8.77 (m, 1 H) 8.57 (dd, J = 7.92, 2.05

Hz, 1 H) 7.59−7.67 (m, 1 H) 7.25−7.31 (m, 2 H) 7.19−7.23 (m, 2

H) 5.56 (s, 2 H) 2.88−3.02 (m, 4 H). MS, m/z 368.0 [M + H]⁺.

General Procedure E: 1-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-

5-yl)methyl)-7-methyl-1H-purin-6(7H)-one (27). 2-(7-Methyl-6-oxo-

6,7-dihydro-1H-purin-1-yl)acetic acid hydrochloride (41f) (0.100 g,

0.409 mmol) was added to a round-bottom ﬂask followed by DMF

(1.6 mL) and CDI (0.073 g, 0.450 mmol). The mixture was stirred at

50 °C for 30 min, (Z)-3-(4-chlorophenyl)-N′-hydroxypropanimida-

mide (50a) (0.81 g, 0.409 mmol) was added, and the temperature was

increased to 100 °C. After 1 h, the mixture was cooled to room

temperature, water and DCM were added, and the layers were

separated. The aqueous portion was extracted with additional DCM,

and the combined organic portions were dried over sodium sulfate,

ﬁltered, and concentrated. The crude material was puriﬁed by silica gel

chromatography, 0−50% 90/10 DCM/MeOH in DCM to provide the

H), 6.83−6.91 (m, 1 H), 4.62 (s, 2 H), 3.79 (s, 3 H), 3.23−3.31 (m, 2

H), 2.69−2.76 (m, 2 H), 2.55 (s, 3 H). MS, m/z 352.0 [M + H]⁺.

N-(4-Chlorophenethyl)-2-(4-oxoquinazolin-3(4H)-yl)acetamide

(10). The title compound was prepared according to general

procedure A using 2-(4-oxoquinazolin-3(4H)-yl)acetic acid (41b)

1

and 2-(4-chlorophenyl)ethanamine as a white solid in 40% yield. H

NMR (400 MHz, DMSO-d₆) δ ppm 8.33−8.38 (m, 1 H), 8.24−8.28

(m, 1 H), 8.12−8.16 (m, 1 H), 7.81−7.87 (m, 1 H), 7.67−7.72 (m, 1

H), 7.52−7.58 (m, 1 H), 7.31−7.36 (m, 2 H), 7.23−7.28 (m, 2 H),

4.58−4.63 (m, 2 H), 4.58−4.63 (m, 2 H), 2.70−2.75 (m, 2 H). MS,

m/z 342.0 [M + H]⁺.

N-(4Cchlorophenethyl)-2-(7-methyl-4-oxoquinazolin-3(4H)-yl)-

acetamide (11). The title compound was prepared according to

general procedure B using 7-methylquinazolin-4(3H)-one (39c) and

2-bromo-N-(4-chlorophenethyl)acetamide (40) as a yellow solid in

1

85% yield. H NMR (400 MHz, DMSO-d₆) δ 8.43 (s, 1H), 8.28 (s,

1H), 8.00 (d, J = 8.4, 1H), 7.54 (s, 1H), 7.43−7.37 (m, 3H), 7.31 (d, J

= 8.4, 2H), 4.63 (s, 2H), 3.39−3.31 (m, 2H), 2.76 (t, J = 7.2, 2H), 2.51

(s, 3H). MS, m/z 355.8 [M + H]⁺.

N-(4-Chlorophenethyl)-2-(6-methyl-4-oxoquinazolin-3(4H)-yl)-

acetamide (12). The title compound was prepared according to

general procedure B using 6-methylquinazolin-4(3H)-one (39d) and

2-bromo-N-(4-chlorophenethyl)acetamide (40) as a yellow solid in

1

80% yield. H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 8.21 (s,

1H), 7.94 (s, 1H), 7.68−7.58 (m, 2H), 7.35−7.24 (m, 4H), 4.60 (s,

2H), 3.34−3.29 (m, 2H), 2.74−2.72 (m, 2H), 2.45 (s, 3H). MS, m/z

355.9 [M + H]⁺.

1

title compound (0.044 g, 24% yield) as a white solid. H NMR (400

MHz, DMSO-d₆) δ ppm 8.45 (s, 1 H), 8.24 (s, 1 H), 7.26−7.31 (m, 2

H), 7.21−7.26 (m, 2 H), 5.53 (s, 2 H), 3.95 (s, 3 H), 2.88−3.02 (m, 4

H). MS, m/z 371.0 [M + H]⁺

N-(4-Chlorophenethyl)-2-(4-oxo-8-(triﬂuoromethyl)quinazolin-

3(4H)-yl)acetamide (14). The title compound was prepared according

to general procedure A using 2-(4-oxo-8-(triﬂuoromethyl)quinazolin-

3(4H)-yl)acetic acid (41c) and 2-(4-chlorophenyl)ethanamine as an

General Procedure F: 7-Methylquinazolin-4(3H)-one (39c). In a

20 mL microwave vial, 2-amino-4-methylbenzoic acid (3.0 g, 19.8

mmol) was dissolved in formamide (7.89 mL, 198 mmol). The

mixture was irradiated at 150 °C for 1 h in the microwave. The

mixture was diluted with water, and the resulting solids were ﬁltered,

rinsed with water, and air-dried to yield the title compound (2.0 g,

1

oﬀ-white solid in 31% yield. H NMR (400 MHz, DMSO-d₆) δ 8.43

(s, 1 H), 8.37−8.45 (m, 2 H), 8.23 (dd, J = 7.53, 0.88 Hz, 1 H), 7.70

(t, J = 7.73 Hz, 1 H), 7.31−7.36 (m, 2 H), 7.24−7.28 (m, 2 H), 4.65

(s, 2 H), 3.30−3.36 (m, 2 H), 2.69−2.77 (m, 2 H). MS, m/z 410.0 [M

+ H]⁺.

N-(4-Chlorophenethyl)-2-(8-ﬂuoro-4-oxoquinazolin-3(4H)-yl)-

acetamide (15). The title compound was prepared according to

general procedure A using 2-(8-ﬂuoro-4-oxoquinazolin-3(4H)-yl)-

acetic acid (41d) and 2-(4-chlorophenyl)ethanamine as an oﬀ-white

solid in 43% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.34−8.41

(m, 1 H), 8.33 (s, 1 H), 7.93−7.98 (m, 1 H), 7.68−7.76 (m, 1 H),

7.51−7.58 (m, 1 H), 7.31−7.37 (m, 2 H), 7.22−7.28 (m, 2 H), 4.63

(s, 2 H), 3.30−3.35 (m, 2 H), 2.69−2.76 (m, 2 H). MS, m/z 360.0 [M

+ H]⁺.

N-(4-Chlorophenethyl)-2-(8-methoxy-4-oxoquinazolin-3(4H)-yl)-

acetamide (16). The title compound was prepared according to

general procedure B using 8-methoxyquinazolin-4(3H)-one (39h) and

2-bromo-N-(4-chlorophenethyl)acetamide (40) as a yellow solid in

6% yield. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 8.35 (t, J = 5.50 Hz,

1 H), 8.21 (s, 1 H), 7.68 (d, J = 8.01 Hz, 1 H), 7.48 (t, J = 7.96 Hz, 1

H), 7.32−7.40 (m, 3 H), 7.26 (d, J = 7.85 Hz, 2 H), 4.61 (s, 2 H), 3.92

(s, 3 H), 3.31 (s, 42 H), 2.72 (t, J = 7.21 Hz, 2 H), 2.50 (br s, 6 H),

0.04 (s, 1 H). MS, m/z 372.0 [M + H]⁺.

1

63% yield) as an oﬀ-white solid. H NMR (400 MHz, DMSO-d₆) δ

12.16 (br s, 1H), 8.05 (s, 1H), 8.00 (d, J = 8.4, 1H), 7.48 (s, 1H), 7.34

(d, J = 8.0, 1H), 2.46 (s, 3H). MS, m/z 161.3 [M + H]⁺.

2-(8-Methyl-4-oxoquinazolin-3(4H)-yl)-N-phenethylacetamide

(5). The title compound was prepared according to general procedure

A using 2-(8-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid (41a) and

1

2-phenylethanamine as a white solid in 70% yield. H NMR (400

MHz, DMSO-d₆) δ ppm 8.42−8.49 (m, 1 H), 8.37 (s, 1 H), 8.02−8.09

(m, 1 H), 7.74−7.80 (m, 1 H), 7.47−7.54 (m, 1 H), 7.34−7.40 (m, 2

H), 7.32 (s, 3 H), 4.70 (s, 2 H), 3.34−3.43 (m, 2 H), 2.76−2.85 (m, 2

H), 2.63 (s, 3 H). MS, m/z 322.0 [M + H]⁺.

N-(4-Methoxyphenethyl)-2-(8-methyl-4-oxoquinazolin-3(4H)-yl)-

acetamide (6). The title compound was prepared according to general

procedure A using 2-(8-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid

(41a) and 2-(4-methoxyphenyl)ethanamine as a white solid in 52%

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 8.31−8.37 (m, 1 H),

8.29 (s, 1 H), 7.95−8.01 (m, 1 H), 7.66−7.72 (m, 1 H), 7.39−7.46

(m, 1 H), 7.10−7.17 (m, 2 H), 6.79−6.88 (m, 2 H), 4.62 (s, 2 H), 3.72

(s, 3 H), 3.23−3.30 (m, 2 H), 2.63−2.71 (m, 2 H), 2.53−2.57 (m, 3

H). MS, m/z 352.0 [M + H]⁺.

N-(4-Chlorophenethyl)-2-(8-cyano-4-oxoquinazolin-3(4H)-yl)-

acetamide (17). The title compound was prepared according to

general procedure B using 4-oxo-3,4-dihydroquinazoline-8-carbonitrile

(39i) and 2-bromo-N-(4-chlorophenethyl)acetamide (40) as a white

N-(3-Methoxyphenethyl)-2-(8-methyl-4-oxoquinazolin-3(4H)-yl)-

acetamide (7). The title compound was prepared according to general

procedure A using 2-(8-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid

(41a) and 2-(3-methoxyphenyl)ethanamine as a white solid in 65%

1

solid in 72% yield. H NMR (400 MHz, DMSO-d₆) δ 8.49 (s, 1 H)

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 8.34−8.39 (m, 1 H),

8.34−8.44 (m, 3 H) 7.70 (t, J = 7.78 Hz, 1 H) 7.31−7.37 (m, 2 H)

7.23−7.29 (m, 2 H) 4.65 (s, 2 H) 3.30−3.36 (m, 2 H) 2.73 (t, J = 7.14

Hz, 2 H). MS, m/z 366.8 [M + H]⁺.

8.29 (s, 1 H), 7.95−8.00 (m, 1 H), 7.67−7.72 (m, 1 H), 7.38−7.47

(m, 1 H), 7.14−7.23 (m, 1 H), 6.74−6.82 (m, 3 H), 4.59−4.64 (m, 2

H), 3.75 (s, 3 H), 3.28−3.35 (m, 2 H), 2.68−2.74 (m, 2 H), 2.54−2.58

(m, 3 H). MS, m/z 352.0 [M + H]⁺.

N-(4-Chlorophenethyl)-2-(4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)-

acetamide (18). The title compound was prepared according to

general procedure B using pyrido[2,3-d]pyrimidin-4(3H)-one (39j)

and 2-bromo-N-(4-chlorophenethyl)acetamide (40) as a yellow solid

N-(2-Methoxyphenethyl)-2-(8-methyl-4-oxoquinazolin-3(4H)-yl)-

acetamide (8). The title compound was prepared according to general

procedure A using 2-(8-methyl-4-oxoquinazolin-3(4H)-yl)acetic acid

(41a) and 2-(2-methoxyphenyl)ethanamine as a white solid in 75%

1

in 64% yield. H NMR (400 MHz, DMSO-d₆) δ 8.99 (dd, J = 4.55,

2.01 Hz, 1 H) 8.55 (dd, J = 7.92, 1.96 Hz, 1 H) 8.47−8.51 (m, 1 H)

8.40 (t, J = 5.67 Hz, 1 H) 7.59 (dd, J = 7.87, 4.55 Hz, 1 H) 7.31−7.38

(m, 2 H) 7.23−7.28 (m, 2 H) 4.64 (s, 2 H) 3.27−3.34 (m, 2 H) 2.69−

2.75 (m, 2 H). MS, m/z 342.8 [M + H]⁺.

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 8.32−8.38 (m, 1 H),

8.28 (s, 1 H), 7.94−8.01 (m, 1 H), 7.67−7.72 (m, 1 H), 7.40−7.46

(m, 1 H), 7.17−7.24 (m, 1 H), 7.11−7.16 (m, 1 H), 6.93−6.98 (m, 1

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N-(4-Chlorophenethyl)-2-(4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)-

acetamide (19). The title compound was prepared according to

general procedure B using pyrido[3,4-d]pyrimidin-4(3H)-one (39k)

and 2-bromo-N-(4-chlorophenethyl)acetamide (40) as an oﬀ-white

solid in 14% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.08−9.12

(m, 1 H), 8.68−8.73 (m, 1 H), 8.41−8.43 (m, 1 H), 8.34−8.41 (m, 1

H), 7.97−8.01 (m, 1 H), 7.31−7.36 (m, 2 H), 7.21−7.29 (m, 2 H),

4.60−4.67 (m, 2 H), 3.30−3.35 (m, 2 H), 2.69−2.76 (m, 2 H). MS,

m/z 343.2 [M + H]⁺.

diazol-5-yl)methanamine (48) as a white solid in 15% yield. ¹H NMR

(400 MHz, DMSO-d₆) δ 8.30−8.35 (m, 1 H) 8.05−8.08 (m, 1 H)

7.25−7.30 (m, 2 H) 7.20−7.25 (m, 2 H) 5.55 (s, 2 H) 4.19 (s, 3 H)

2.96−3.02 (m, 2 H) 2.90−2.96 (m, 2 H). MS, m/z 371.0 [M + H]⁺.

3-((3-(2-(6-Chloropyridin-3-yl)ethyl)-1,2,4-oxadiazol-5-yl)-

methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (30). The title compound

was prepared according to general procedure E using 2-(4-oxopyrido-

[2,3-d]pyrimidin-3(4H)-yl)acetic acid hydrochloride (41e) and (Z)-3-

(6-chloropyridin-3-yl)-N′-hydroxypropanimidamide (50b) as a yellow

1

3-((5-(4-Chlorophenethyl)-1,3,4-oxadiazol-2-yl)methyl)pyrido-

[2,3-d]pyrimidin-4(3H)-one (22). A vial was charged with 2-(4-

oxopyrido[2,3-d]pyrimidin-3(4H)-yl)acetic acid (41d) (80 mg, 0.390

mmol), 3-(4-chlorophenyl)propanehydrazide (51) (93 mg, 0.468

mmol), EtOAc (0.260 mL), DMF (0.520 mL), and triethylamine

(0.163 mL, 1.170 mmol). 1-Propanephosphonic acid cyclic anhydride,

50 wt % solution in ethyl acetate (0.744 mL, 1.170 mmol), was added,

and the mixture was stirred overnight at 90 °C. Water and EtOAc were

added, and the layers were separated. The organic portion was dried

over sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by silica gel chromatography, using a gradient of 0−50% 90/

10 DCM/MeOH in DCM to provide the desired compound (25 mg,

solid in 85% yield. H NMR (400 MHz, DMSO-d₆) δ 9.03 (dd, J =

1.96, 4.60 Hz, 1H), 8.76 (s, 1H), 8.57 (dd, J = 2.05, 7.92 Hz, 1H),

8.20−8.30 (m, 1H), 7.71 (dd, J = 2.54, 8.22 Hz, 1H), 7.63 (dd, J =

4.55, 7.97 Hz, 1H), 7.37 (d, J = 8.22 Hz, 1H), 5.51−5.62 (m, 2H),

2.92−3.07 (m, 4H). MS, m/z 369.0 [M + H]⁺.

3-((3-(2-(4-Chlorophenyl)-2-hydroxyethyl)-1,2,4-oxadiazol-5-yl)-

methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (31). The title compound

was prepared according to general procedure E using 2-(4-oxopyrido-

[2,3-d]pyrimidin-3(4H)-yl)acetic acid hydrochloride (41e) and (Z)-3-

(4-chlorophenyl)-N′,3-dihydroxypropanimidamide (50c) as an oﬀ-

white solid in 23% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (dd, J

= 2.05, 4.60 Hz, 1H), 8.76 (s, 1H), 8.58 (dd, J = 2.05, 7.92 Hz, 1H),

7.60−7.67 (m, 1H), 7.28−7.37 (m, 4H), 5.60 (d, J = 4.79 Hz, 1H),

5.56 (s, 2H), 4.92 (td, J = 5.39, 7.70 Hz, 1H), 2.94−3.01 (m, 2H). MS,

m/z 384.0 [M + H]⁺.

1

0.068 mmol, 18% yield) as an oﬀ white solid. H NMR (400 MHz,

DMSO-d₆) δ 9.02 (dd, J = 2.01, 4.55 Hz, 1H), 8.69−8.76 (m, 1H),

8.52−8.60 (m, 1H), 7.57−7.68 (m, 1H), 7.21−7.30 (m, 4H), 5.48 (s,

2H), 3.12−3.22 (m, 2H), 2.95−3.04 (m, 2H). MS, m/z 367.9 [M +

H]⁺.

3-((3-(2-(4-Chlorophenyl)-2-ﬂuoroethyl)-1,2,4-oxadiazol-5-yl)-

methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (32). In a sealed tube, 3-

((3-(2-(4-chlorophenyl)-2-hydroxyethyl)-1,2,4-oxadiazol-5-yl)-

methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (31) (0.012 mg, 0.031

mmol) was dissolved in DCM (0.063 mL). The solution was cooled

to 0 °C, and (diethylamino)sulfur triﬂuoride (0.012 mL, 0.094 mmol)

was added dropwise. The mixture was allowed to warm to RT and

stirred for 1 h. The reaction was carefully quenched with sodium

bicarbonate and was extracted into DCM. The organic portion was

washed with water and brine, dried with sodium sulfate, ﬁltered

through a fritted funnel, and concentrated. The crude product was

puriﬁed with silica gel chromatography using a gradient of 25−70%

DCM:MeOH(90:10)/DCM to provide the desired compound as a

6-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)pyrimido-

[4,5-c]pyridazin-5(6H)-one (23). The title compound was prepared

according to general procedure D using 3-aminopyridazine-4-

carboxylic acid and (3-(4-chlorophenethyl)-1,2,4-oxadiazol-5-yl)-

methanamine (48) as an oﬀ-white solid in 25% yield. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.58 (d, J = 5.18 Hz, 1 H) 8.83−8.91 (m, 1

H) 8.35 (d, J = 5.18 Hz, 1 H) 7.25−7.32 (m, 2 H) 7.18−7.24 (m, 2 H)

5.52−5.62 (m, 2 H) 2.86−3.03 (m, 4 H). MS, m/z 368.8 [M + H]⁺.

3-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)pyrimido-

[4,5-d]pyrimidin-4(3H)-one (24). The title compound was prepared

according to general procedure D using 4-aminopyrimidine-5-

carboxylic acid and (3-(4-chlorophenethyl)-1,2,4-oxadiazol-5-yl)-

methanamine (48) as a white solid in 51% yield. ¹H NMR (400

MHz, DMSO-d₆) δ 9.56 (s, 1 H) 9.50 (s, 1 H) 8.97 (s, 1 H) 7.25−

7.35 (m, 2 H) 7.18−7.24 (m, 2 H) 5.58 (s, 2 H) 2.85−3.06 (m, 4 H).

MS, m/z 368.8 [M + H]⁺.

1

racemic mixture (7 mg, 0.018 mmol, 58.0% yield) as a tan solid. H

NMR (400 MHz, DMSO-d₆) δ 9.04 (dd, J = 1.96, 4.60 Hz, 1H), 8.77

(s, 1H), 8.57 (dd, J = 2.05, 7.92 Hz, 1H), 7.61−7.66 (m, 1H), 7.39−

7.51 (m, 5H), 5.84−6.03 (m, 1H), 5.59 (s, 2H), 3.41−3.53 (m, 1H),

3.34−3.41 (m, 1H). MS, m/z 386.0 [M + H]⁺.

3-((5-(2-(4-Chlorophenyl)propyl)-1,2,4-oxadiazol-3-yl)methyl)-

pyrido[2,3-d]pyrimidin-4(3H)-one (33). The title compound was

prepared according to general procedure C using 3-(4-chlorophenyl)-

butanoic acid (45) and (Z)-N′-hydroxy-2-(4-oxopyrido[2,3-d]-

pyrimidin-3(4H)-yl)acetimidamide (43a) as an oﬀ-white solid in

26% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.02 (dd, J = 2.05, 4.60

Hz, 1H), 8.71 (s, 1H), 8.54 (dd, J = 2.01, 7.87 Hz, 1H), 7.62 (dd, J =

4.55, 7.97 Hz, 1H), 7.25 (s, 4H), 5.34 (s, 2H), 3.20−3.30 (m, 3H),

1.23 (d, J = 6.55 Hz, 3H). MS, m/z 382.0 [M + H]⁺.

3-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)pteridin-

4(3H)-one (25). The title compound was prepared according to

general procedure D using 3-amino-2-pyrazinecarboxylic acid and (3-

(4-chlorophenethyl)-1,2,4-oxadiazol-5-yl)methanamine (48) as a light

1

yellow solid in 70% yield. H NMR (400 MHz, DMSO-d₆) δ 9.07−

9.11 (m, 1 H) 8.92−8.96 (m, 1 H) 8.84 (s, 1 H) 7.25−7.30 (m, 2 H)

7.18−7.25 (m, 2 H) 5.60 (s, 2 H) 2.96−3.03 (m, 2 H) 2.90−2.96 (m,

2 H). MS, m/z 369.0 [M + H]⁺.

3-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)-5-

methylpyrido[2,3-d]pyrimidin-4(3H)-one (26). The title compound

was prepared according to general procedure D using 2-amino-4-

methylnicotinic acid and (3-(4-chlorophenethyl)-1,2,4-oxadiazol-5-

(E)-3-((5-(4-Chlorostyryl)-1,2,4-oxadiazol-3-yl)methyl)pyrido[2,3-

d]pyrimidin-4(3H)-one (34). The title compound was prepared

according to general procedure C using (E)-3-(4-chlorophenyl)acrylic

acid (46) and (Z)-N′-hydroxy-2-(4-oxopyrido[2,3-d]pyrimidin-3(4H)-

1

yl)methanamine (48) as an oﬀ-white solid in 20% yield. H NMR

(400 MHz, DMSO-d₆) δ 8.79 (d, J = 4.79 Hz, 1 H) 8.68−8.72 (m, 1

H) 7.41 (dd, J = 4.84, 0.83 Hz, 1 H) 7.24−7.29 (m, 2 H) 7.19−7.24

(m, 2 H) 5.50 (s, 2 H) 2.89−3.03 (m, 4 H) 2.76 (s, 3 H). MS, m/z

381.9 [M + H]⁺.

1

yl)acetimidamide (43a) as an oﬀ-white solid in 43% yield. H NMR

(400 MHz, DMSO-d₆) δ 8.99−9.05 (m, 1H), 8.75−8.82 (m, 1H),

8.52−8.59 (m, 1H), 7.85−7.89 (m, 1H), 7.82−7.86 (m, 2H), 7.62 (dd,

J = 4.60, 7.92 Hz, 1H), 7.48−7.54 (m, 2H), 7.41 (d, J = 16.53 Hz, 1H),

5.45 (s, 2H). MS, m/z 366.0 [M + H]⁺.

(E)-3-((5-(2-(4-Chlorophenyl)prop-1-en-1-yl)-1,2,4-oxadiazol-3-

yl)methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (35). The title com-

pound was prepared according to general procedure C using (E)-3-

(4-chlorophenyl)but-2-enoic acid (47) and (Z)-N′-hydroxy-2-(4-

oxopyrido[2,3-d]pyrimidin-3(4H)-yl)acetimidamide (43a) as an oﬀ-

white solid in 42% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.00−9.05

(m, 1H), 8.77−8.83 (m, 1H), 8.54−8.59 (m, 1H), 7.69−7.77 (m, 2H),

7.58−7.66 (m, 1H), 7.45−7.54 (m, 2H), 6.91−6.96 (m, 1H), 5.47 (s,

2H), 2.60 (d, J = 1.27 Hz, 3H). MS, m/z 380.0 [M + H]⁺.

1-((5-(4-Chlorophenethyl)-1,2,4-oxadiazol-3-yl)methyl)-7-meth-

yl-1H-purin-6(7H)-one (28). The title compound was prepared

according to general procedure C using 3-(4-chlorophenyl)propionic

acid (44) and (Z)-N′-hydroxy-2-(7-methyl-6-oxo-6,7-dihydro-1H-

purin-1-yl)acetimidamide (43b) as an oﬀ-white solid in 32% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.39−8.44 (m, 1H), 8.19−8.24 (m,

1H), 7.22−7.30 (m, 4H), 5.31−5.40 (m, 2H), 3.95 (s, 3H), 3.18−3.28

(m, 2H), 2.97−3.05 (m, 2H). MS, m/z 371.0 [M + H]⁺.

6-((3-(4-Chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)-1-meth-

yl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (29). The title compound

was prepared according general procedure D using 4-amino-2-methyl-

2H-pyrazole-3-carboxylic acid and (3-(4-chlorophenethyl)-1,2,4-oxa-

K

DOI: 10.1021/acs.jmedchem.6b00039

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(E)-1-((5-(2-(4-Chlorophenyl)prop-1-en-1-yl)-1,2,4-oxadiazol-3-

yl)methyl)-7-methyl-1H-purin-6(7H)-one (36). The title compound

was prepared according to general procedure c using (E)-3-(4-

chlorophenyl)but-2-enoic acid (47) and (Z)-N′-hydroxy-2-(7-methyl-

6-oxo-6,7-dihydro-1H-purin-1-yl)acetimidamide (43b) as a white solid

in 42% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (s, 1H), 8.22 (s,

1H), 7.74 (d, J = 8.61 Hz, 2H), 7.51 (d, J = 8.71 Hz, 2H), 6.94 (d, J =

0.98 Hz, 1H), 5.45 (s, 2H), 3.96 (s, 3H), 2.61 (s, 3H). MS, m/z 383.0

[M + H]⁺.

Pyrido[2,3-d]pyrimidin-4(3H)-one (39j). The title compound was

prepared according to general procedure F using 2-aminonicotinic acid

1

as a tan solid in 49% yield. H NMR (400 MHz, DMSO-d₆) δ 12.40

(br s, 1H), 8.95 (dd, J = 2.05, 4.60 Hz, 1H), 8.50 (dd, J = 2.05, 7.92

Hz, 1H), 8.31 (s, 1H), 7.55 (dd, J = 4.60, 7.92 Hz, 1H). MS, m/z

147.9 [M + H]⁺.

7-Methyl-1H-purin-6(7H)-one (39l). A pressure bottle was charged

with 6-chloro-7-methyl-7H-purine²¹(2.12 g, 12.58 mmol), dioxane

(50 mL), and hydrochloric acid (2 M in dioxane, 12.58 mL, 25.2

mmol), and the reaction was stirred at 90 °C for 2 h. The mixture was

concentrated to provide the title compound as the hydrochloride salt

(E)-1-((3-(2-(4-Chlorophenyl)prop-1-en-1-yl)-1,2,4-oxadiazol-5-

yl)methyl)-7-methyl-1H-purin-6(7H)-one (37). The title compound

was prepared according to general procedure E using 2-(7-methyl-6-

oxo-6,7-dihydro-1H-purin-1-yl)acetic acid hydrochloride (41f) and

(1Z,2E)-3-(4-chlorophenyl)-N′-hydroxybut-2-enimidamide (50d) as

1

(2.4 g, 12.9 mmol, 100% yield) as a tan solid. H NMR (400 MHz,

DMSO-d₆) δ 8.61 (s, 1H), 8.10 (s, 1H), 4.01 (s, 3H). MS, m/z 151.0

[M + H]⁺.

1

2-Bromo-N-(4-chlorophenethyl)acetamide (40). To a solution of

bromoacetic acid (4.46 g, 32.1 mmol) in 40.0 mL of EtOAc at 0 °C

was added 1-propanephosphonic acid cyclic anhydride, 50 wt % in

EtOAc (30.7 mL, 48.2 mmol). To the resulting mixture was added a

solution of 2-(4-chlorophenyl)ethanamine (4.50 mL, 32.1 mmol) and

triethylamine (8.96 mL, 64.3 mmol) in EtOAc (20.0 mL). The

resulting mixture was stirred at room temperature overnight. The

mixture was washed twice with water and brine and was dried over

Na₂SO₄. The solvents were removed in vacuo. The crude material was

absorbed onto a plug of silica gel and puriﬁed by chromatography,

eluting with 0% to 50% EtOAc/heptane, to provide the title

compound (5.1 g, 18.44 mmol, 57% yield) as a white solid. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.32 (br s, 1H), 7.30−7.39 (m, 2H),

7.20−7.27 (m, 2H), 3.81 (s, 2H), 3.25−3.31 (m, 2H), 2.71 (t, J = 7.14

Hz, 2H). MS, m/z 275.7/277.7 [M + H]⁺.

an oﬀ-white solid in 45% yield. H NMR (400 MHz, DMSO-d₆) δ

8.49 (s, 1H), 8.25 (s, 1H), 7.65−7.68 (m, 2H), 7.45−7.49 (m, 2H),

6.70 (d, J = 1.27 Hz, 1H), 5.61 (s, 2H), 3.96 (s, 3H), 3.32 (s, 3H). MS,

m/z 383.1 [M + H]⁺.

8-Methylquinazolin-4(3H)-one (39a). The title compound was

prepared according to general procedure F using 2-amino-3-

methylbenzoic acid as an oﬀ-white solid in 29% yield. ¹H NMR

(400 MHz, DMSO-d₆) δ 12.19 (br s, 1H), 8.10 (s, 1H), 7.96 (dd, J =

0.98, 7.92 Hz, 1H), 7.64−7.70 (m, 1H), 7.36−7.43 (m, 1H), 2.52−

2.56 (m, 3H). MS, m/z 161.2 [M + H]⁺.

6-Methylquinazolin-4(3H)-one (39d). The title compound was

prepared according to general procedure F using 2-amino-5-

methylbenzoic acid as a tan solid in 57% yield. ¹H NMR (400

MHz, DMSO-d₆) δ 12.16 (s, 1H), 8.05−8.00 (m, 1H), 7.91 (s, 1H),

7.67−7.53 (m, 2H), 2.44 (s, 3H). MS, m/z 161.0 [M + H]⁺.

5-Methylquinazolin-4(3H)-one (39e). The title compound was

prepared according to general procedure F using 2-amino-6-

methylbenzoic acid as a white solid in 44% yield. ¹H NMR (400

MHz, DMSO-d₆) δ 11.99 (br s, 1H), 7.98 (s, 1H), 7.62 (t, J = 7.57 Hz,

1H), 7.46 (d, J = 7.73 Hz, 1H), 7.25 (d, J = 7.34 Hz, 1H), 2.77 (s, 3H).

MS, m/z.161.0 [M + H]⁺.

8-(Triﬂuoromethyl)quinazolin-4(3H)-one (39f). The title com-

pound was prepared according to general procedure F using 2-

amino-3-(triﬂuoromethyl)benzoic acid. Water and EtOAc were added,

and the layers were separated. The organic portion was dried over

sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by silica gel chromatography, 0−100% EtOAc/heptane to

provide the desired compound in 26% yield as a yellow solid. MS, m/z

215.0 [M + H]⁺.

2-(8-Methyl-4-oxoquinazolin-3(4H)-yl)acetic Acid (41a). A pres-

sure bottle was charged with 8-methylquinazolin-4(3H)-one (39a)

(2.88 g, 17.98 mmol), potassium carbonate (3.73 g, 27.0 mmol), DMF

(71.9 mL), and methyl 2-bromoacetate (2.04 mL, 21.58 mmol), and

the mixture was stirred at 50 °C for 1.5 h. The mixture was poured

into water, and the resulting solids were ﬁltered, washed with water,

and air-dried to provide the title compound (3.501 g, 15.08 mmol,

1

84% yield) as an oﬀ-white solid. H NMR (400 MHz, DMSO-d₆) δ

8.36−8.41 (m, 1H), 7.95−8.01 (m, 1H), 7.70−7.75 (m, 1H), 7.42−

7.49 (m, 1H), 4.84 (s, 2H), 3.71 (s, 3H), 2.56 (s, 3H). MS, m/z 233.0

[M + H]⁺. A round-bottom ﬂask was charged with methyl 2-(8-

methyl-4-oxoquinazolin-3(4H)-yl)acetate (3.5 g, 15.07 mmol), THF

(22.61 mL), water (7.54 mL), and LiOH (1.805 g, 75 mmol), and the

mixture was stirred at RT for 1 h. The mixture was brought to pH 2 by

addition of 2 N HCl and was extracted twice with EtOAc. The

combined organic portions were dried over sodium sulfate, ﬁltered,

and concentrated. Solids remained in the aqueous layer, which were

ﬁltered, washed with water, and dried. The solids from the organic and

aqueous portions were combined and dried under high vacuum to

provide the desired compound (3.01 g, 13.79 mmol, 92% yield) as a

8-Fluoroquinazolin-4(3H)-one (39g). The title compound was

prepared according to general procedure F using 2-amino-3-

1

ﬂuorobenzoic acid as a tan solid in 57% yield. H NMR (400 MHz,

DMSO-d₆) δ ppm 12.27−12.52 (m, 1 H), 8.14 (s, 1 H), 7.93 (dt, J =

8.00, 1.04 Hz, 1 H), 7.68 (ddd, J = 10.66, 8.02, 1.37 Hz, 1 H), 7.51 (td,

J = 8.02, 4.79 Hz, 1 H). MS, m/z 165.0 [M + H]⁺.

8-Methoxyquinazolin-4(3H)-one (39h). The title compound was

prepared according to general procedure F using 2-amino-3-

methoxybenzoic acid as a tan solid in 77% yield. MS, m/z 177.0 [M

+ H]⁺.

4-Oxo-3,4-dihydroquinazoline-8-carbonitrile (39i). 8-Bromoqui-

nazolin-4(3H)-one was prepared according to general procedure F

using 2-amino-3-bromobenzoic acid as a yellow solid in 54% yield. ¹H

NMR (400 MHz, DMSO-d₆) δ 12.48 (br s, 1H), 8.21 (s, 1H), 8.08−

8.17 (m, 2H), 7.39−7.47 (m, 1H). MS, m/z 225.0/227.0 [M + H]⁺. A

pressure bottle was charged with 8-bromoquinazolin-4(3H)-one (1.50

g, 6.67 mmol), Pd(PPh₃)₄(0.770 g, 0.667 mmol), zinc cyanide (2.348

g, 20.00 mmol), and DMF (26.7 mL). The bottle was sealed, and the

mixture was heated at 100 °C for 24 h. The mixture was poured into

water, and the resulting solids were ﬁltered, washed with water, and

dried. This material was triturated in DCM, ﬁltered, washed with

DCM, and dried. Solids also precipitated out of the aqueous ﬁltrate

and were ﬁltered, washed with water, and dried. The solids were

combined to provide the desired compound as a white solid. The

material was used without further puriﬁcation. MS, m/z 172.0 [M +

H]⁺.

1

tan solid. H NMR (400 MHz, DMSO-d₆) δ 13.19 (br s, 1H), 8.30−

8.45 (m, 1H), 7.94−8.04 (m, 1H), 7.67−7.76 (m, 1H), 7.38−7.50 (m,

1H), 4.66−4.81 (m, 2H), 2.55 (s, 3H). MS, m/z 219.0 [M + H]⁺.

2-(4-Oxoquinazolin-3(4H)-yl)acetic Acid (41b). A vial was charged

with quinazolin-4(3H)-one (39b) (1.00 g, 6.84 mmol), K₂CO₃(1.419

g, 10.26 mmol), DMF (13.68 mL), and methyl 2-bromoacetate (0.696

mL, 7.53 mmol), and the mixture was heated at 50 °C for 4 h. Water

and EtOAc were added, and the layers were separated. The organic

portion was dried over sodium sulfate, ﬁltered, and concentrated. The

crude material was dried under high vacuum to provide methyl 2-(4-

oxoquinazolin-3(4H)-yl)acetate as a white solid (1.3 g, 5.96 mmol,

1

87% yield). H NMR (400 MHz, DMSO-d₆) δ ppm 8.35−8.39 (m, 1

H), 8.12−8.17 (m, 1 H), 7.84−7.90 (m, 1 H), 7.69−7.74 (m, 1 H),

7.55−7.61 (m, 1 H), 4.85 (s, 2 H), 3.71 (s, 3 H). MS, m/z 219.0 [M +

H]⁺. A round-bottom ﬂask was charged with methyl 2-(4-

oxoquinazolin-3(4H)-yl)acetate (500 mg, 2.291 mmol), THF (6.8

mL), water (0.31 mL), and LiOH (549 mg, 22.91 mmol). The mixture

was stirred overnight at RT. The mixture was brought to acidic pH

with 2 N HCl, and EtOAc was added. The layers were separated, and

the organic portion was dried over sodium sulfate, ﬁltered, and

concentrated to provide the title compound as a white solid (410 mg,

L

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2.00 mmol, 88% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm

13.14−13.23 (m, 1 H), 8.34−8.37 (m, 1 H), 8.12−8.18 (m, 1 H),

7.83−7.89 (m, 1 H), 7.69−7.73 (m, 1 H), 7.52−7.61 (m, 1 H), 4.70−

4.75 (m, 2 H). MS, m/z 205.0 [M + H]⁺.

2-(7-Methyl-6-oxo-6,7-dihydro-1H-purin-1-yl)acetic Acid Hydro-

chloride (41f). A vial was charged with 7-methyl-1H-purin-6(7H)-one

(39l) (180 mg, 1.199 mmol), potassium carbonate (199 mg, 1.439

mmol), tert-butyl bromoacetate (0.175 mL, 1.199 mmol), and DMF

(2.40 mL). The mixture was heated at 50 °C for 6 h. Water and EtOAc

were added, and the layers were separated. The organic portion was

dried with sodium sulfate, ﬁltered, and concentrated. The crude

material was puriﬁed by silica gel chromatography, 0−40% 90/10

DCM/MeOH in DCM, to provide tert-butyl 2-(7-methyl-6-oxo-6,7-

dihydro-1H-purin-1-yl)acetate (110 mg, 0.416 mmol, 34.7% yield) as a

2-(4-Oxo-8-(triﬂuoromethyl)quinazolin-3(4H)-yl)acetic Acid

(41c). A vial was charged with 8-(triﬂuoromethyl)quinazolin-4(3H)-

one (39f) (0.200 mg, 0.93 mmol), potassium carbonate (0.19 mg, 1.40

mmol), methyl 2-bromoacetate (0.11 mL, 1.12 mmol), and DMF (1.9

mL). The mixture was heated at 50 °C for 2 h. Water and EtOAc were

added, and the layers were separated. The organic portion was dried

over sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by silica gel chromatography, using 15−70% EtOAc/heptane

to provide methyl 2-(4-oxo-8-(triﬂuoromethyl)quinazolin-3(4H)-yl)-

1

white foamy solid. H NMR (400 MHz, DMSO-d₆) δ 8.22−8.27 (m,

1H), 8.16−8.21 (m, 1H), 4.66−4.76 (m, 2H), 3.92−4.01 (m, 3H),

1.39−1.48 (m, 9H). MS, m/z 265.0 [M + H]⁺. Tert-butyl 2-(7-methyl-

6-oxo-6,7-dihydro-1H-purin-1-yl)acetate (110 mg, 0.416 mmol) was

dissolved in concentrated HCl in a vial and stirred at RT. After 30 min,

the mixture was concentrated and the title compound (86 mg, 0.413

1

acetate (0.160 g, 0.559 mmol, 59.9% yield) as a light yellow solid. H

NMR (400 MHz, DMSO-d₆) δ 8.52 (s, 1H), 8.40−8.47 (m, 1H), 8.25

(d, J = 7.63 Hz, 1H), 7.72 (t, J = 7.92 Hz, 1H), 4.88 (s, 2H), 3.68−

3.77 (m, 3H).MS, m/z 287.0 [M + H]⁺. Methyl 2-(4-oxo-8-

(triﬂuoromethyl)quinazolin-3(4H)-yl)acetate (0.160 g, 0.559 mmol)

and LiOH (0.062 g, 2.80 mmol) were added to water (0.28 mL) and

THF (0.84 mL). The mixture was stirred at room temperature for 1 h.

The mixture was brought to pH 2 by addition of 2 N HCl and was

extracted twice with EtOAc. The combined organics were dried over

sodium sulfate, ﬁltered, and concentrated to yield (130 mg, 0.478

mmol, 85% yield) as an oﬀ-white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 13.32 (br s, 1H), 8.49−8.55 (m, 1H), 8.43 (dd, J = 0.98, 8.02 Hz,

1H), 8.19−8.29 (m, 1H), 7.66−7.78 (m, 1H), 4.69−4.85 (m, 2H).

MS, m/z 273.0 [M + H]⁺.

1

mmol, 99% yield) was isolated as an oﬀ-white solid. H NMR (400

MHz, DMSO-d₆) δ 8.52 (s, 1H), 8.34−8.38 (m, 1H), 4.76 (s, 2H),

3.97−4.02 (m, 3H). MS, m/z 209.0 [M + H]⁺.

2-(4-Oxopyrido[2,3-d]pyrimidin-3(4H)-yl)acetonitrile (42a). A vial

was charged with pyrido[2,3-d]pyrimidin-4(3H)-one (39j) (150 mg,

1.019 mmol), potassium carbonate (211 mg, 1.529 mmol),

bromoacetonitrile (0.071 mL, 1.070 mmol), and DMF (4.08 mL).

The reaction was stirred at 50 °C for 1 h. Water and EtOAc were

added, and the layers were separated. The organic portion was dried

with sodium sulfate, ﬁltered, and concentrated, to provide the desired

1

compound (96 mg, 51% yield). H NMR (400 MHz, DMSO-d₆) δ

9.01 (dd, J = 2.05, 4.60 Hz, 1H), 8.62−8.65 (m, 1H), 8.60 (dd, J =

2.05, 7.92 Hz, 1H), 7.63 (dd, J = 4.60, 7.92 Hz, 1H), 5.08−5.15 (m,

2H). MS, m/z 187.0 [M + H]⁺.

2-(8-Fluoro-4-oxoquinazolin-3(4H)-yl)acetic Acid (41d). A vial

was charged with 8-ﬂuoroquinazolin-4(3H)-one (39g) (0.200 g, 1.22

mmol), potassium carbonate (0.25 g, 1.83 mmol), methyl 2-

bromoacetate (0.14 mL, 1.46 mmol), and DMF (2.4 mL). The

mixture was heated at 50 °C for 2 h. The mixture was poured into

water, and the resulting solids were ﬁltered, washed with water, and

dried to provide methyl 2-(8-ﬂuoro-4-oxoquinazolin-3(4H)-yl)acetate

2-(7-Methyl-6-oxo-6,7-dihydro-1H-purin-1-yl)acetonitrile (42b).

7-Methyl-1H-purin-6(7H)-one hydrochloride (39l) (900 mg, 4.82

mmol), potassium carbonate (1666 mg, 12.06 mmol), DMF (19.300

mL), and bromoacetonitrile (0.336 mL, 4.82 mmol) were stirred at 55

°C for 2 h. Upon cooling to RT, water and DCM were added. The

layers were extracted, and the aqueous portion was extracted again

with DCM. The combined organic portions were dried over sodium

sulfate, ﬁltered, and concentrated to provide the title compound (260

1

(0.170 g, 0.720 mmol, 59.1% yield) as a light yellow solid. H NMR

(400 MHz, DMSO-d₆) δ 8.52 (s, 1H), 8.40−8.47 (m, 1H), 8.25 (d, J =

7.63 Hz, 1H), 7.72 (t, J = 7.92 Hz, 1H), 4.88 (s, 2H), 3.68−3.77 (m,

3H).MS, m/z 287.0 [M + H]⁺. Methyl 2-(8-ﬂuoro-4-oxoquinazolin-

3(4H)-yl)acetate (0.170 g, 0.720 mmol) and LiOH (0.086 g, 3.60

mmol) were added to water (0.36 mL) and THF (1.08 mL). The

mixture was stirred at room temperature for 1 h. The mixture was

brought to pH 2 by addition of 2 N HCl and was extracted twice with

EtOAc. The combined organics were dried over sodium sulfate,

ﬁltered, and concentrated to yield the title compound (110 mg, 0.495

mmol, 69% yield) as an oﬀ-white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 13.14−13.35 (m, 1 H), 8.42 (s, 1 H), 7.90−8.04 (m, 1 H),

7.68−7.81 (m, 1 H), 7.41−7.61 (m, 1 H), 4.76 (s, 2 H). MS, m/z

223.0 [M + H]⁺.

1

mg, 1.374 mmol, 29% yield) as a white solid. H NMR (400 MHz,

DMSO-d₆) δ ppm 8.34−8.37 (m, 1 H), 8.20−8.26 (m, 1 H), 5.09−

5.12 (m, 2 H), 3.96−4.00 (s, 3 H). MS, m/z 190.0 [M + H]⁺.

(Z)-N′-Hydroxy-2-(4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)-

acetimidamide (43a). A vial was charged with 2-(4-oxopyrido[2,3-

d]pyrimidin-3(4H)-yl)acetonitrile (42a) (96 mg, 0.516 mmol), EtOH

(2.06 mL), and hydroxylamine (0.126 mL, 2.063 mmol). The

heterogeneous mixture was stirred at 80 °C for 1 h. Upon cooling,

the solids were ﬁltered, washed with EtOH, and dried under high

vacuum to provide the title compound (72 mg, 0.328 mmol, 63.7%

yield) as a rust-colored solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.16−

9.27 (m, 1H), 8.92−9.01 (m, 1H), 8.52−8.57 (m, 1H), 8.48−8.52 (m,

1H), 7.53−7.61 (m, 1H), 5.64−5.77 (m, 2H), 4.57−4.71 (m, 2H).

MS, m/z 219.9 [M + H]⁺.

2-(4-Oxopyrido[2,3-d]pyrimidin-3(4H)-yl)acetic Acid Hydrochlor-

ide (41e). A vial was charged with pyrido[2,3-d]pyrimidin-4(3H)-one

(39j) (5.00 g, 34.0 mmol), potassium carbonate (7.04 g, 51.0 mmol),

tert-butyl bromoacetate (5.94 mL, 40.8 mmol), and DMF (34 mL).

The mixture was heated at 70 °C for 1 h. Water and EtOAc were

added, and the layers were separated. The organic portion was dried

with sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by silica gel chromatography, 25−100% 90/10 DCM/MeOH

in DCM, to provide tert-butyl 2-(4-oxopyrido[2,3-d]pyrimidin-3(4H)-

(Z)-N′-Hydroxy-2-(7-methyl-6-oxo-6,7-dihydro-1H-purin-1-yl)-

acetimidamide (43b). A vial was charged with 2-(7-methyl-6-oxo-6,7-

dihydro-1H-purin-1-yl)acetonitrile (42b) (260 mg, 1.374 mmol),

EtOH (5498 μL), and hydroxylamine (337 μL, 5.50 mmol). The

reaction mixture was heated at 80 °C for 2 h. Upon cooling to RT,

water was added, and the solids were ﬁltered, washed with water, and

dried. The title compound (145 mg, 0.653 mmol, 47.5% yield) was

1

yl)acetate (7.2 g, 27.6 mmol, 81.0% yield) as a light yellow solid. H

1

NMR (400 MHz, DMSO-d₆) δ 9.00 (dd, J = 2.01, 4.55 Hz, 1H), 8.58

(s, 1H), 8.56 (dd, J = 2.05, 7.92 Hz, 1H), 7.61 (dd, J = 4.60, 7.92 Hz,

1H), 4.74 (s, 2H), 1.41−1.47 (m, 9H). MS, m/z 261.8 [M + H]⁺. tert-

Butyl 2-(4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl)acetate (7.2 g, 27.6

mmol) was dissolved in dichloromethane (55.1 mL). Hydrochloric

acid (2 N in dioxane, 11.31 mL, 138.0 mmol) was added, and the

reaction was stirred at RT for 30 min. The reaction mixture was

concentrated in vacuo to provide the desired product (4.9 g, 74%) as a

isolated as a tan solid. H NMR (400 MHz, DMSO-d₆) δ ppm 9.17−

9.21 (m, 1 H), 8.14−8.19 (m, 2 H), 5.59−5.66 (m, 2 H), 4.59−4.64

(m, 2 H), 3.93−3.98 (m, 3 H). MS, m/z 223.2 [M + H]⁺.

3-(4-Chlorophenyl)butanoic Acid (45). To a round-bottom ﬂask

was added a spatula tip of Raney nickel (slurry in water) followed by a

solution of (E)-ethyl 3-(4-chlorophenyl)but-2-enoate (prepared in

step 1, intermediate 47) (700 mg, 3.12 mmol) dissolved in 6 mL of

EtOH. The ﬂask was evacuated and ﬁlled with hydrogen. The reaction

was stirred at 20 °C for 18 h under hydrogen atmosphere. The mixture

was passed through a pad of Celite and rinsed with EtOH. The ﬁltrate

was concentrated, and the crude material was puriﬁed via reverse phase

1

light yellow solid. H NMR (400 MHz, DMSO-d₆) δ 9.01 (dd, J =

2.05, 4.60 Hz, 1H), 8.62 (s, 1H), 8.60 (dd, J = 2.05, 7.92 Hz, 1H), 7.64

(dd, J = 4.60, 7.92 Hz, 1H), 4.77 (s, 2H). MS, m/z 206.0 [M + H]⁺.

M

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chromatography using a gradient of 50−100% acetonitrile (0.1%

TFA):water (0.1% TFA). The product containing fractions were

combined and brought to pH 8 with saturated NaHCO_3.The aqueous

portion was extracted into ethyl acetate, dried with sodium sulfate,

ﬁltered through a fritted funnel, and concentrated to yield ethyl 3-(4-

chlorophenyl)butanoate (160 mg, 0.706 mmol, 23% yield) as a light

7.36 (m, 2H), 7.22−7.30 (m, 2H), 3.91 (s, 2H), 2.94−3.02 (m, 4H),

2.00−2.09 (m, 2H). MS, m/z 238.0 [M + H]⁺.

3-(6-Chloropyridin-3-yl)propanenitrile (49b). A round-bottom

ﬂask was charged with diethyl cyanomethylphosphonate (1.50 g,

8.48 mmol) in THF (14.1 mL). NaH (60% dispersion, 0.339 g, 8.48

mmol) was added, and the mixture was stirred at RT for 10 min until

gas evolution ceased. After cooling to 0 °C, 6-chloronicotinaldehyde

(1.00 g, 7.06 mmol) was added, and the reaction was allowed to warm

to RT. EtOAc and water were added, and the layers were separated.

The organic portion was dried over sodium sulfate, ﬁltered, and

concentrated. The crude material was triturated in diethyl ether,

ﬁltered, washed with diethyl ether, and dried to provide (E)-3-(6-

chloropyridin-3-yl)acrylonitrile (500 mg, 3.04 mmol, 43.0% yield) as

1

yellow oil. H NMR (400 MHz, DMSO-d₆) δ 7.24−7.36 (m, 4H),

3.94−4.01 (m, 2H), 3.13−3.20 (m, 1H), 2.58 (dd, J = 4.21, 7.53 Hz,

2H), 1.17−1.21 (m, 3H), 1.09 (t, J = 7.09 Hz, 3H). MS, m/z 227.0 [M

+ H]⁺. A round-bottom ﬂask was charged with ethyl 3-(4-

chlorophenyl)butanoate (160 mg, 0.706 mmol), THF (1.06 mL),

water (0.353 mL), and LiOH (85 mg, 3.53 mmol). The mixture was

stirred at RT for 2 h, brought to pH 2 by addition of 2 N HCl, and

extracted twice with EtOAc. The combined organic portions were

dried over sodium sulfate, ﬁltered, and concentrated to yield the title

compound (120 mg, 0.604 mmol, 86% yield) as an oﬀ-white solid. ¹H

NMR (400 MHz, DMSO-d₆) δ 12.03 (s, 1H), 7.23−7.36 (m, 4H),

3.06−3.22 (m, 1H), 2.47−2.50 (m, 2H), 1.19 (d, J = 6.94 Hz, 3H).

MS, m/z 199.0 [M + H]⁺.

1

an oﬀ-white solid. H NMR (400 MHz, chloroform-d) δ 8.48 (d, J =

2.54 Hz, 1H), 7.76 (dd, J = 2.54, 8.31 Hz, 1H), 7.42 (d, J = 24 Hz,

1H), 7.40 (d, J = 1.76 Hz, 1H), 5.97 (d, J = 16.73 Hz, 1H). MS, m/z

165.0 [M + H]⁺. A vial was charged with (E)-3-(6-chloropyridin-3-

yl)acrylonitrile (300 mg, 1.823 mmol), pyridine (5.47 mL), and

MeOH (1.82 mL). NaBH₄(103 mg, 2.73 mmol) was added, and the

mixture was heated at 70 °C for 2 h. EtOAc and saturated NH₄Cl were

added, and the layers were separated. The organic portion was dried

over sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by silica gel chromatography, 0−50% EtOAc/heptane, to give

the title compound (90 mg, 0.540 mmol, 29.6% yield) as a yellow oil.

MS, m/z 167.0 [M + H]⁺.

3-(4-Chlorophenyl)-3-hydroxypropanenitrile (49c). A round-bot-

tom ﬂask was charged with 3-(4-chlorophenyl)-3-oxopropanenitrile

(1.0 g, 5.57 mmol) and EtOH (11.14 mL). The solution was cooled to

0 °C, and sodium borohydride (0.421 g, 11.14 mmol) was added. After

stirring at 0 °C for 1 h, the reaction was quenched with saturated

ammonium chloride and the ice bath was removed. The reaction was

extracted into dichloromethane, washed with water and brine, dried

over sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by silica gel chromatography, using a gradient of 10−75% 90/

10 DCM/MeOH in DCM, to provide the desired compound (0.80 g,

4.40 mmol, 79% yield) as a colorless oil. ¹H NMR (400 MHz, DMSO-

d₆) δ 7.39−7.47 (m, 4H), 6.03 (d, J = 4.50 Hz, 1H), 4.86−4.96 (m,

1H), 2.86−2.95 (m, 1H), 2.78−2.86 (m, 1H). MS, m/z 204.0 [M +

Na]⁺.

(E)-3-(4-Chlorophenyl)but-2-enenitrile (49d). A vial was charged

with diethyl cyanomethylphosphonate (2.52 mL, 14.23 mmol) and

THF (25.9 mL). NaH (60% dispersion) (0.569 g, 14.23 mmol) was

added, and the reaction was stirred at room temperature for 10 min.

4′-Chloroacetophenone (1.681 mL, 12.94 mmol) was added and the

reaction stirred for 1 h. EtOAc and saturated NH₄Cl were added, and

the layers were separated. The aqueous phase was extracted with

EtOAc, and the combined organic portions were dried over MgSO₄,

ﬁltered, and concentrated. The crude material was puriﬁed via silica gel

column chromatography, eluting with 0−50% EtOAc/heptane. The

product containing fractions were combined and concentrated in

vacuo to yield the desired compound (2.25 g, 98% yield). MS, m/z

178.2 [M + H]⁺.

(E)-3-(4-Chlorophenyl)but-2-enoic Acid (47). In a 100 mL round-

bottom ﬂask NaH (60% dispersion in mineral oil) (168 mg, 4.20

mmol) were dissolved in 9 mL of THF and triethyl phosphonoacetate

(0.798 mL, 3.56 mmol) was added dropwise. In a separate ﬂask, 4′-

chloroacetophenone (500 mg, 3.23 mmol) was dissolved in 4 mL of

THF and added quickly to the phosphonate mixture. The reaction was

stirred for 30 min at RT, cooled to 0 °C, and diluted with methanol.

Saturated ammonium chloride was slowly added until a precipitate

formed. The solids were ﬁltered and rinsed with ethyl acetate. Water

was added to the ﬁltrate, and the layers were separated. The organic

portion was dried with sodium sulfate, ﬁltered, and concentrated. The

crude material was puriﬁed with reverse phase chromatography using a

gradient of 50−100% acetonitrile (0.1% TFA):water (0.1% TFA). The

product containing fractions were brought to pH 8 with saturated

NaHCO_3.The aqueous portion was extracted into ethyl acetate, dried

with sodium sulfate, ﬁltered through a fritted funnel, and concentrated

to yield (E)-ethyl 3-(4-chlorophenyl)but-2-enoate (220 mg, 0.979

mmol 30.3% yield) as a colorless oil. ¹H NMR (400 MHz, chloroform-

d) δ 7.38−7.46 (m, 2H), 7.32−7.38 (m, 2H), 6.09−6.15 (m, 1H),

4.16−4.27 (m, 2H), 2.52−2.61 (m, 3H), 1.33 (t, J = 7.14 Hz, 3H).

MS, m/z 225.0 [M + H]⁺. A round-bottom ﬂask was charged with (E)-

ethyl 3-(4-chlorophenyl)but-2-enoate (100 mg, 0.445 mmol), THF

(668 μL), water (223 μL), and LiOH (53.3 mg, 2.225 mmol), and the

mixture was stirred at RT for 17 h. The mixture was brought to pH 2

by addition of 2 N HCl and was extracted twice with EtOAc. The

combined organic portions were dried over sodium sulfate, ﬁltered,

and concentrated to yield the desired compound as an oﬀ-white solid.

MS, m/z 197.0 [M + H]⁺.

(3-(4-Chlorophenethyl)-1,2,4-oxadiazol-5-yl)methanamine (48).

A vial was charged with N-Boc-glycine (441 mg, 2.52 mmol), (Z)-3-

(4-chlorophenyl)-N′-hydroxypropanimidamide (50a) (500 mg, 2.52

mmol), triethylamine (1.75 mL, 12.58 mmol), and EtOAc (5.03 mL).

1-Propanephosphonic acid cyclic anhydride, 50 wt % solution in ethyl

acetate (4.00 mL, 6.29 mmol) was added, and the mixture was stirred

at 80 °C for 5 h. Water and EtOAc were added, and the layers were

separated. The organic portion was dried over sodium sulfate, ﬁltered,

and concentrated. The crude material was puriﬁed by silica gel

chromatography (Isolera), 0−50% EtOAc/heptane, to provide tert-

butyl ((3-(4-chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)carbamate

(487 mg, 1.442 mmol, 57.3% yield) as a colorless oil. ¹H NMR

(400 MHz, chloroform-d) δ 7.24−7.29 (m, 2H), 7.12−7.17 (m, 2H),

5.14 (br s, 1H), 4.49−4.63 (m, 2H), 2.95−3.13 (m, 4H), 1.48 (s, 9H).

MS, m/z 360.0 [M + Na]⁺. A vial was charged with tert-butyl ((3-(4-

chlorophenethyl)-1,2,4-oxadiazol-5-yl)methyl)carbamate (480 mg,

1.421 mmol), DCM (2.84 mL) and TFA (1.10 mL, 14.21 mmol),

and the mixture was stirred at RT overnight. The mixture was

concentrated, dissolved in DCM, and washed with saturated NaHCO₃.

The organic portion was dried over sodium sulfate, ﬁltered, and

concentrated. The title compound (290 mg, 1.220 mmol, 86% yield)

(Z)-3-(4-Chlorophenyl)-N′-hydroxypropanimidamide (50a). A

pressure bottle was charged with 3-(4-chlorophenyl)propionitrile

(49a) (2.0 mL, 12.08 mmol), EtOH (24.15 mL), and hydroxylamine

(3.19 mL, 48.3 mmol). The mixture was stirred at 80 °C for 1.5 h.

Upon cooling to RT, water was added and the mixture was allowed to

stand. The mixture was poured onto ice, which caused precipitation.

The solids were ﬁltered, washed with water, and dried to provide the

1

title compound (2.23 g, 11.23 mmol, 93% yield) as a white solid. H

NMR (400 MHz, DMSO-d₆) δ 8.70−8.76 (m, 1H), 7.29−7.35 (m,

2H), 7.20−7.27 (m, 2H), 5.41 (s, 2H), 2.73−2.83 (m, 2H), 2.17−2.25

(m, 2H). MS, m/z 199.0 [M + H]⁺.

(Z)-3-(6-Chloropyridin-3-yl)-N′-hydroxypropanimidamide (50b).

A vial was charged with 3-(6-chloropyridin-3-yl)propanenitrile (49b)

(90 mg, 0.540 mmol), EtOH (2.16 mL), and hydroxylamine (0.132

mL, 2.161 mmol), and the mixture was stirred at 45 °C for 24 h. Water

and EtOAc were added, the layers were separated, and the organic

portion was dried over sodium sulfate, ﬁltered, and concentrated to

give the desired product (93 mg, 0.466 mmol, 86% yield) as a slightly

1

was isolated as a tan solid. H NMR (400 MHz, DMSO-d₆) δ 7.30−

N

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1

yellow oil. H NMR (400 MHz, DMSO-d₆) δ 8.70−8.81 (m, 1H),

calculated using the formula: % control (POC) = (compound −

average low)/(average high − average low) × 100. For IC₅₀

determination, data were ﬁtted to a four-parameter equation (y = A

+ ((B − A)/(1 + ((x/C)^∧D))), where A is the minimum y (POC)

value, B is the maximum y (POC), C is the x (compound

concentration) at the point of inﬂection, and D is the slope factor).

⁴⁵Ca²⁺Assays (rTRPA1, hTRPA1, antagonist and agonist mode).

These assays were run according to the published procedures.^22,23

hTRPV1, hTRPV4, rTRPV3, rTRPM8 Assays. These assays were run

according to the published procedure.²³

8.19−8.32 (m, 1H), 7.71 (dd, J = 2.54, 8.22 Hz, 1H), 7.36−7.46 (m,

1H), 5.43 (s, 2H), 2.73−2.90 (m, 2H), 2.18−2.32 (m, 2H). MS, m/z

200.0 [M + H]⁺.

(Z)-3-(4-Chlorophenyl)-N′,3-dihydroxypropanimidamide (50c). A

vial was charged with 3-(4-chlorophenyl)-3-hydroxypropanenitrile

(49c) (200 mg, 1.101 mmol), EtOH (2.20 mL), and hydroxylamine

(0.270 mL, 4.40 mmol). The solution was stirred at 80 °C for 1 h.

Upon cooling to RT, the EtOH was evaporated under reduced

pressure. Water and EtOAc were added, and the layers were separated.

The organic portion was dried over sodium sulfate, ﬁltered, and

concentrated to yield the desired product (172 mg, 0.801 mmol, 72.8%

Antagonist against Methylglyoxal (MG) Assay. Cells were

generated and maintained as described for the TRPA1 calcium ﬂux

assays. On the day of assay, culture media was removed and cells were

incubated for 10 min at RT with 50 μL of compound in compound

dilution buﬀer (HBSS containing 1 mM HEPES + 0.1 mg/mL BSA] at

ﬁnal concentrations (2.0 nM to 40 μM, 1:3 ratio) prior to the addition

50 μL of agonist (MG at ﬁnal concentration 2.74 mM) and radioactive

calcium (ﬁnal concentration10 μCi/mL) in assay buﬀer (Ham’s F-12

containing 15 mM HEPES + 0.1% BSA), followed by another 3 min

incubation at RT. The reaction mixture was aspirated, and cells were

washed three times with phosphate buﬀer saline (PBS) containing 0.1

mg/mL BSA. Radioactivity was measured using a TopCount

microplate scintillation counter. The activation of TRPA1 was

measured by the cellular uptake of radioactive calcium. The agonist

(MG)-induced ⁴⁵Ca²⁺uptake was considered as 100%, and the wells

with radioactive assay buﬀer without agonist were taken as 0%.

Antagonist against Hypotonicity Assay. Cells were generated and

maintained as described for the TRPA1 calcium ﬂux assays. On the day

of assay, culture media was removed and cells were incubated for 10

min at RT with 20 μL of compound in normal assay buﬀer (Ham’s F-

12 containing 15 mM HEPES + 0.1% BSA) at ﬁnal concentrations

(2.0 nM to 40 μM, 1:3 ratio) prior to the addition 80 μL of Hypo-

Osmolarity buﬀer (50 mOsmol/L, diluted assay buﬀer, at a ﬁnal

osmilarity 100 mOsmol) with radioactive calcium (ﬁnal concen-

tration10 μCi/mL), followed by another 3 min incubation at RT. The

reaction mixture was aspirated, and cells were washed three times with

phosphate buﬀer saline (PBS) containing 0.1 mg/mL BSA. Radio-

activity was measured using a TopCount microplate scintillation

counter. The activation of TRPA1 was measured by cellular uptake of

radioactive calcium. The hypo-Osmolarity buﬀer-induced uptake was

considered as 100%, and wells with normal radioactive assay buﬀer

(300 mOsmol/L) were taken as 0%.

1

yield) as a light yellow waxy solid. H NMR (400 MHz, DMSO-d₆) δ

8.76 (s, 1H), 7.32−7.37 (m, 4H), 5.35−5.43 (m, 2H), 4.81−4.91 (m,

1H), 3.12−3.19 (m, 1H), 2.26−2.34 (m, 1H), 2.18−2.26 (m, 1H).

MS, m/z 215.0 [M + H]⁺.

(1Z,2E)-3-(4-Chlorophenyl)-N′-hydroxybut-2-enimidamide (50d).

(E)-3-(4-chlorophenyl)but-2-enenitrile (49d) (2.25 g, 12.67 mmol)

was added to a microwave vial followed by EtOH (50.7 mL) and

hydroxylamine (0.776 mL, 12.67 mmol)). The vial was capped and

heated to 100 °C in the microwave reactor for 1 h. The solvent was

removed in vacuo to provide the title comound (2.45 g, 92% yield) as

1

an oﬀ-white solid. H NMR (400 MHz, DMSO-d₆) δ 9.39−9.42 (m,

1H), 7.47−7.52 (m, 2H), 7.39−7.44 (m, 2H), 6.03−6.12 (m, 1H),

5.49 (s, 2H), 2.28 (d, J = 1.37 Hz, 3H). MS, m/z 211.2 [M + H]⁺.

3-(4-Chlorophenyl)propanehydrazide (51). A pressure bottle was

charged with methyl 3-(4-chlorophenyl)propanoate (1.05 g, 5.29

mmol), EtOH (10.57 mL) and hydrazine hydrate (2.62 mL, 52.9

mmol). The bottle was sealed, and the mixture was heated at 100 °C

for 17 h. A small amount of water was added, and the mixture was

allowed to stand at RT. Crystals formed, which were ﬁltered, washed

with water, and dried. The title compound (685 mg, 3.45 mmol, 65.2%

1

yield) as isolated as a white solid. H NMR (400 MHz, DMSO-d₆) δ

ppm 8.89−8.98 (m, 1 H), 7.28−7.35 (m, 2 H), 7.23 (s, 2 H), 4.10−

4.17 (m, 2 H), 2.73−2.84 (m, 2 H), 2.26−2.34 (m, 2 H). MS, m/z

199.0 [M + H]⁺.

TRPA1 Calcium Flux Cell Assays. The generation of CHO cell lines

stably expressing human and rat TRPA1 in tetracycline-inducible T-

REx expression systems (Gibco-Invitrogen, Carlsbad, CA) was

previously described.²³Both human and rat TRPA1 cell lines were

maintained in Ham’s F-12 nutrient mix (Gibco-Invitrogen) containing

10% tetracycline-screened fetal bovine serum (Hyclone, Logan, UT),

1% penicillin/streptomycin/glutamine solution (Gibco-Invitrogen), 10

mg/mL blasticidin S HCl (Gibco-Invitrogen), 250 mg/mL zeocin

(Gibco-Invitrogen), and 400 mg/mL geneticin (Gibco-Invitrogen).

Intracellular calcium ﬂux was measured with the Fluo-4 NW (no-

wash) ﬂuorescent calcium-sensing dye (Molecular Probes, Eugene,

OR) and the FLIPR Tetra ﬂuorescence kinetic plate reader (Molecular

Devices, Sunnyvale CA). Twenty-four hours before dye-loading, cells

were seeded 20000 cells/well in 384-well ﬂat clear-bottom black assay

plates (Costar, Corning, NY) in culture media supplemented with 0.5

mg/mL tetracycline (Corning, Manassas, VA). Fluo-4 NW dye and

water-soluble probenecid (Gibco-Invitrogen) were reconstituted with

assay buﬀer (Hank’s Balanced Salt Solution with 20% HEPES, Gibco-

Invitrogen). Culture media was removed before the addition of

reconstituted dye to the cell plates. Cells were incubated with the dye

at 37 °C for 30 min before equilibrating at room temperature for an

additional 30 min. Two additions were conducted on the FLIPR Tetra

to measure in-sequence agonist and antagonist activity. Test

compounds were added in the ﬁrst addition, and agonist activity

data were collected for 2 min. An EC₉₀concentration (3 μM for

human TRPA1 and 35 μM for rat TRPA1) of TRPA1 agonist allyl

isothiocyanate (AITC) (Fluka-Sigma-Aldrich, St. Louis, MO) was then

added, and antagonist activity was measured for a further 2 min of data

collection. Data were analyzed with Screener (Genedata AG, Basel,

Switzerland) data analysis software. The amount of signal generated in

the presence of compounds versus that in the presence of low control

(DMSO vehicle alone) and high control (for agonists, a saturating

concentration of AITC, 15 μM for humanTRPA1, and 200 μM for rat

TRPA1; for antagonists, an EC₉₀concentration of AITC) was

CYP Inhibition IC₅₀. Inhibition of CYP3A4 and 2D6 was

determined as described.²⁴

Transport across MDCK Cells. Transport studies to determine

apparent permeability and eﬄux ratios [eﬄux ratio = P_app,B→A

/

P_app,A→B] were performed in MDCK vector control or MDR1-

transfected MDCK cells as described.²⁵

AITC Target Engagement Assay. The AITC target engagement

assay was run according to the published procedure.²⁶

ASSOCIATED CONTENT

■

S

* Supporting Information

. The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b00039.

TRPA1 potency data with standard deviations, CEREP

panel data for compound 27, and computational method

for predicting π-stacking interaction of 27 (PDF)

SMILES data for 27 (CSV)

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: laurie.schenkel@amgen.com. Phone: 617-444-5244.

Notes

The authors declare no competing ﬁnancial interest.

O

DOI: 10.1021/acs.jmedchem.6b00039

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(12) Kremeyer, B.; Lopera, F.; Cox, J. J.; Momin, A.; Rugiero, F.;

Marsh, S.; Woods, C. g.; Jones, N. G.; Paterson, K. J.; Fricker, F. R.;

villegas, A.; Acosta, N.; Pineda-Trujillo, N. g.; Ramirez, J. D.; Zea, J.;

Burley, M.-W.; Bedoya, G.; Bennett, D. L.-H.; Wood, J. N.; Ruiz-

Linares, A. A gain-of-function mutation in TRPA1 causes familial

episodic pain syndrome. Neuron 2010, 66, 671−680.

(13) Wheeler, S. E.; Houk, K. N. Substituent effects in the benzene

dimer are due to direct interactions of the substituents with the

unsubstituted benzene. J. Am. Chem. Soc. 2008, 130, 10854−10855.

(14) Predicted interaction energies for compounds 9 and 14−19

were taken from ref 14using the value for the corresponding

substituted phenyl ring. The predicted interaction energy for 27 was

calculated using the same method. See Supporting Information.

(15) Geometries were minimized by using B3LYP/6-31G* and

overlaid using the quinazolinone cores.

ACKNOWLEDGMENTS

■

The authors thank Loren Berry for plasma protein binding

measurements, Virginia Berry and Xuhai Be for bioanalysis of

the PK and PD studies, Jingzhou Liu for metabolite

identiﬁcation studies, Mary Wells for pharmacokinetic and

drug metabolism studies, and Margaret Chu-Moyer for

manuscript input.

ABBREVIATIONS USED

■

CFA, complete Freund’s adjuvant; CDI, carbonyldiimidiazole;

DAST, diethylaminosulfur triﬂuoride; EDC, 1-ethyl-3-(3-

(dimethylamino)propyl)carbodiimide; FLIPR, ﬂuorometric

imaging plate reader; PBS, phosphate-buﬀered saline; SIF,

simulated intestinal ﬂuid; T3P, propylphosphonic anhydride

(16) See Supporting Information.

(17) Dib-Hajj, S. D.; Yang, Y.; Black, J. A.; Waxman, S. G. The Na_v1.7

sodium channel: from molecule to man. Nat. Rev. Neurosci. 2013, 14,

49−62.

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DOI: 10.1021/acs.jmedchem.6b00039

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Optimization of a Novel Quinazolinone-Based Series of Transient Receptor Potential A1 (TRPA1) Antagonists Demonstrating Potent in Vivo Activity

DOI: 10.1021/acs.jmedchem.6b00039

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