Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain

ARTICLE

pubs.acs.org/jmc

Identification of a Potent, State-Dependent Inhibitor of Nav1.7

with Oral Efficacy in the Formalin Model of Persistent Pain

Howard Bregman,*^,†Loren Berry,^‡John L. Buchanan,^†April Chen,^‡Bingfan Du,^†Elma Feric,^§

Markus Hierl,^§Liyue Huang,^‡David Immke,^{^}Brett Janosky,^‡Danielle Johnson,^§Xingwen Li,^‡

Joseph Ligutti,^{^}Dong Liu,^{^}Annika Malmberg,^§David Matson,^§Jeﬀ McDermott,^§Peter Miu,^||

Hanh Nho Nguyen,^†Vinod F. Patel,^†Daniel Waldon,^‡Ben Wilenkin,^§Xiao Mei Zheng,^†Anruo Zou,^{^}

Stefan I. McDonough,^§and Erin F. DiMauro^†

^†Department of Chemistry Research and Discovery, ^‡Department of Pharmacokinetics and Drug Metabolism, and ^§Department of

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

Department of Lead Discovery, and ^{^}Department of Neuroscience, Amgen, Inc., One Amgen Center Drive, Thousand Oaks,

California 91320, United States

S Supporting Information

b

ABSTRACT:

Clinical human genetic studies have recently identiﬁed the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel

Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structureꢀactivity relationships for a novel series of

2,4-diaminotriazines that inhibit hNav1.7. Optimization eﬀorts culminated in compound 52, which demonstrated pharmacokinetic

properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that

of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and

exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium

channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent eﬃcacy in the formalin model

of pain.

’ INTRODUCTION

control point for pain. Clinical genetic studies by several groups

have found that loss of function truncation mutations of SCN9A

result in the complete abrogation of the ability to perceive pain,

without additional major physiological abnormalities.^5ꢀ8Con-

versely, gain of function mutations in SCN9A that increase

channel opening cause chronic pain in the absence of injury.^9ꢀ11

Accordingly, much eﬀort has been focused on generating potent

inhibitors of Nav1.7 that lack inhibitory eﬀects on other members

of the sodium channel family.^12ꢀ22This functional selectivity

may be achieved via a combination of subtype selectivity for

Nav1.7 and state dependence, a property by which compounds

Voltage-gated sodium ion channels have long been recognized

as potentially attractive drug targets for the treatment of chronic

pain, including disorders such as ﬁbromyalgia, painful diabetic

neuropathy, osteoarthritis, and cancer-related pain.¹Existing

antiepileptic or antiarrhythmic drugs that are sodium channel

inhibitors have some eﬃcacy against several forms of chronic

pain,²and intramuscular administration of tetrodotoxin is eﬀec-

tive for relief of pain associated with cancer or chemotherapy.³

These treatments, however, are limited by side eﬀects that are

presumably due to the inhibition of the sodium channels that

inﬂuence other brain, heart, or muscle function. Among the nine

members of the voltage-gated sodium channel family (Nav1.1ꢀ

Nav1.9),⁴the Nav1.7 (SCN9A) gene stands out as a critical

Received: January 7, 2011

Published: June 02, 2011

r

2011 American Chemical Society

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Scheme 1. Preparation of Diaminotriazines 4^a

^aReagents: (a) DIEA, DMF, 0ꢀ20 ꢀC; (b) DIEA, DMF, NHR³R⁴, 20 ꢀC. R¹, R²= H, Me; R³, R⁴= varied.

Scheme 2. Preparation of Diaminopyrimidines 7, 9, 12^a

^aReagents: (a) N-(3-aminophenyl)acetamide, DIEA, i-PrOH, 0ꢀ20 ꢀC, 31%; (b) 1,2,3,4-tetrahydroisoquinoline, DIEA, i-PrOH, 0ꢀ20 ꢀC, 88% (8),

82% (11); (c) 1,2,3,4-tetrahydroisoquinoline, TFA, i-PrOH, 90 ꢀC, 50%; (d) N-(3-aminophenyl)acetamide, TFA, i-PrOH, 90 ꢀC, 80% (9), 34% (12).

preferentially inhibit the inactivated states of sodium channels

thought to have greater prevalence in diseased tissue such as the

hyperexcitable neurons driving chronic pain.^23,24Here we report

the characterization, structureꢀactivity relationship (SAR), and

optimization of a series of Nav1.7 inhibitors derived from a novel

small molecule scaﬀold identiﬁed using a high-throughput elec-

trophysiology screen.²⁵This eﬀort produced a tool compound

with novel biophysical properties that was eﬀective in the rat

formalin model of pain and represents a step toward identifying

new therapeutics for the control of pain via subtype-speciﬁc state-

dependent inhibition of sodium ion channels.

wide range of anilines and amines with the order of addition

being paramount. For example, reaction of primary or secondary

amines directly with 2,4-dichlorotriazine led to a mixture of

mono and bis-substitution adducts.

The diaminopyrimidine derivatives 7, 9, and 12 were prepared

from the respective dichloropyrimidine precursors in analogous

two-step processes (Scheme 2). Substitution at the more elec-

trophilic 4-position of 2,4-dichloropyrimidine (5) by 3-aminoa-

cetanilide proceeded smoothly under general basic conditions,

providing intermediate 2-chloropyrimidine 6 which upon heat-

ing with 1,2,3,4-tetrahydroisoquinoline in the presence of tri-

ﬂuoroacetic acid (TFA) aﬀorded pyrimidine 7. By reversal of

the sequence of nucleophilic additions, pyrimidine 9 was isolated

via the intermediacy of chloropyrimidine 8. Starting with 4,6-

dichloropyrimidine (10), an analogous sequence of basic and

subsequent acidic S_NAr reactions aﬀorded 4,6-disubstituted

derivative 12.

The synthesis of corresponding N-methylated derivative 15

proceeded via the intermediacy of aryl bromide 14 which was

prepared employing a one pot double S_NAr procedure (Scheme 3).

Sequential exposure of 2,4-dichlorotriazine (1) with 3-bromo-N-

methylaniline (13) and 1,2,3,4-tetrahydroisoquinoline, respec-

tively, in N,N-dimethylformamide (DMF) furnished 14, which

’ CHEMISTRY

The preparation of diaminotriazine 4 proceeded via two

sequential nucleophilic aromatic substitution reactions (S_NAr)

(Scheme 1). Reaction of a 3-aminoacetanilide (2) with 2,4-

dichlorotriazine (1) in the presence of N,N-diisopropylethyla-

mine (DIEA) led to the clean formation of monochlorotriazine

3,²⁶which could be either isolated chromatographically prior to

the subsequent nucleophilic aromatic substitution or treated in

one pot directly with primary or secondary amines at ambient

temperature leading to the formation of desired product (4).^27,28

This protocol was found to be general and reproducible for a

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Scheme 3. Preparation of N-Modiﬁed or Substituted Triazines 15 and 18^a

^aReagents: (a) (i) DIEA, 3-bromo-N-methylaniline, DMF, 0ꢀ20 ꢀC; (ii) tetrahydroisoquinoline, 20 ꢀC, 25%; (b) acetamide, X-Phos, Cs₂CO₃,

Pd₂(dba)₃, 1,4-dioxane, 110 ꢀC, 35%; (c) 1,2,3,4-tetrahydroisoquinoline, DIEA, DMF, 0ꢀ20 ꢀC, 54%; (d) NaH, DMF, 20 ꢀC, 75%.

model of persistent pain. Accordingly, compounds were screened

by automated high throughput patch-clamp electrophysiology

(PatchXpress) on live cells expressing Nav1.7. We utilized a

voltage protocol that enabled assessment of compound state

dependence with a holding voltage set to inactivate approxi-

mately 20% of the channels. Our screen identiﬁed lead com-

pound 24 as a potent, state-dependent inhibitor having structural

features unique among known sodium channel antagonists.

Manual whole-cell patch-clamp experiments on Nav1.7 con-

ﬁrmed the initial PatchXpress results, with IC₅₀= 0.9 μM for

Nav1.7 with partially inactivated channels (20% inactivated) and

IC₅₀> 30 μM recorded on noninactivated channels.³¹Addition-

ally, compound 24 exhibited modest selectivity over the cardiac

sodium channel Nav1.5 (IC₅₀= 5.4 μM) and the hERG (human

ether a-go-go-related gene) potassium channel (IC₅₀= 3.8 μM)

(Figure 1). A major challenge we confronted was the poor

pharmacokinetic (PK) proﬁle of lead compound 24, which had

high clearance (4.0 (L/h)/kg). Although compound 24 had

good permeability, it possessed poor solubility, an issue to be

addressed en route to identifying an agent suitable for oral

dosing (Figure 1). We embarked on a comprehensive SAR

evaluation around 24 with the goals of improving potency and

PK properties.

Initial studies were focused on modiﬁcation of the core and

aminophenylacetamide moieties. It was determined that most

point mutations led to an erosion of potency (see Table 1). All

three pyrimidine derivatives constituting single point nitrogen to

carbonꢀhydrogen atom replacements were prepared (7, 9, 12).

Of this set only 4,6-pyrimidine derivative 12 was equipotent with

triazine 24, while the 2,4 pyrimidine analogues (7, 9) were

weakly active. Methylation of the aniline or amide nitrogen (15

and 25, respectively) or replacement of the aniline NH with

oxygen (18) resulted in a dramatic loss of potency. On the basis

of these results, SAR evaluations of the tetrahydroisoquinoline

portion of the molecule were performed with the triazine and

3-aminophenylacetamide groups locked as the core and left-

hand-side fragments, respectively. The 4,6-pyrimidine core would

be revisited once the rest of the molecule had been optimized and

will be discussed later.

Scheme 4. Preparation of Piperidine Ether Substituted Dia-

minotriazines 23^a

^a(a) (i) NaH, DMF, 0ꢀ20 ꢀC; (ii) RX; (b) TFA, DCM, 20 ꢀC; (c) 21,

DIEA, DMF, 20 ꢀC.

was coupled with acetamide under BuchwaldꢀHartwig condi-

tions to yield triazine 15. Derivative 18, in which the aniline

nitrogen is replaced with an oxygen, was prepared by reaction of

chlorotriazine 16 with the sodium alkoxide of N-(3-hydroxyphe-

nyl)acetamide (17) in DMF at ambient temperature.

The preparation of modiﬁed benzyloxypiperidines 21 was

amenable to a parallel synthetic approach (Scheme 4). Depro-

tonation of N-Boc-4-hydroxypiperidine (19) with sodium hy-

dride in DMF followed by alkylation with benzyl halides aﬀorded

N-protected piperidines 20. TFA mediated cleavage of the tert-

butoxycarbonyl (Boc) group of 20 aﬀorded secondary amines 21

which were puriﬁed by a catch and release protocol utilizing

strong cation exchange (SCX-2) chromatography.²⁹Piperidines

21 were treated with DIEA in DMF in the presence of chloro-

triazine 22 at ambient temperature to provide desired com-

pounds 23.

’ RESULTS AND DISCUSSION

Nonselective sodium channel inhibitors such as mexiletine

that are used oﬀ-label as third-line treatments for pain display

preferential inhibition of inactivated channel states. This form of

state-dependent potency is likely the feature that confers their

modest therapeutic window.^23,30We sought a state-dependent

small molecule inhibitor of Nav1.7 with suﬃcient potency and

pharmacokinetic properties to achieve target coverage with oral

dosing in order to determine if it would be eﬀective in a rat formalin

We suspected that the electron rich and conformationally rigid

tetrahydroquinoline moiety was a liability in terms of oxidative

metabolism and solubility and therefore sought to identify the

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Table 1. SAR: Variations to the Core and Left-Hand-Side

Polar Moieties

Figure 1. In vitro activity and pharmacokinetic properties of com-

pound 24.

minimal critical pharmacophore. Upon examination of the right-

hand-side (RHS) SAR (see Table 2) it was determined that

truncation of the tetrahydroisoquinoline was unfavorable. For

example, compounds containing amino (26) and unsubstituted

piperidine (27) groups had poor activity. The introduction of

various small substituents at the 4-position did not provide a

boost in potency. For example, methyl substitution (28) and

methoxy (29) and hydrophilic groups such as a primary carbox-

amide (30) were not tolerated. However, we were delighted to

learn that submicromolar potency could be realized with the

appropriate larger hydrophobic modiﬁcation at the 4-position of

the piperidine. For example, 4-phenyl substituted compound

(31) exhibited submicromolar potency. Introducing polar substi-

tution in addition to hydrophobicity in this region of chemical

space also led to a drop in potency (33 and 34). However, potency

was retained when one- or two-atom homologations of phenyl-

piperidine (31) were made, as in phenoxypiperidine (35) and

benzyloxypiperidine (36), respectively. Interestingly, replace-

ment of the oxygen atom of 35 with a methylene (32) led to a

signiﬁcant reduction in potency.

The potencies and microsomal stabilities of 35 and 36 were

maintained relative to 24, and the solubility properties were

markedly improved (see Table 3). The observed improvement in

solubility is possibly due to the additional elements of rotational

freedom present in the phenoxypiperidine and benzyloxypiper-

idine moieties or the addition of a heteroatom relative to the

tetrahydroisoquinoline. Compounds exhibiting acceptable po-

tency (<2 μM) and low to moderate in vitro intrinsic clearance

(CL_int) in rat liver microsomes (RLM) (<200 (μL/min)/mg)

were assessed in rat PK studies (see Table 3). Both phenoxypi-

peridine (35) and benzyloxypiperidine (36) derivatives had low

clearance in rats (0.59 and 0.45 L/(h kg), respectively), and

3

moderate to good oral bioavailability (F) (F = 52% and 19% for

compounds 35 and 36, respectively). Because of their signiﬁcantly

improved PK properties relative to lead 24, compounds 35 and

36 were chosen for further SAR examination with the goal of

increasing target coverage by a combination of potency and PK

improvements.

Because the 2-aminotriazine motif is a hinge-binder element

present in many kinase inhibitors,²⁷aminotriazine 36 was screened

at 1 μM against a panel of 48 kinases and was found to inhibit six

kinases by greater than 50% (Abl, 82%; Fyn, 85%; Lyn, 74%; Src,

81%; Mst2, 76%; BTK, 54%). It was envisioned that introduction

of a substituent adjacent to the linker binder element may

disrupt the kinase inhibitory activity via a combination of steric

and/or conformational eﬀects. Accordingly, compound 37 was

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Table 2. SAR: Variations to the Right-Hand-Side Amine

Moiety

prepared, featuring a methyl group at the 2-position of the

aminophenylacetamide moiety. This perturbation led to a re-

duction in kinase inhibitory activity, with 37 inhibiting no kinase

by greater than 10% from the same panel of 48 kinases. We found

that, in general, introduction of a 2-methyl substituent retained

potency and microsomal stability (see Table 4 for a comparison

of des-methyl (36, 31, 35) and methylated analogues (37ꢀ39)).

Therefore, optimization of lead compounds 37 and 39 was

initiated with N-(3-amino-2-methylphenyl)acetamide ﬁxed, and

further piperidine ether modiﬁcations were explored using a

focused library synthetic approach.

The SAR of the aryloxypiperidine series derived from lead 39

closely paralleled that of the benzyloxypiperidine series derived

from lead 37 in terms of substitution about the aryl ring of the

piperidine ether moiety. Therefore, only the SAR for the benzyl-

oxypiperidine series will be reported in detail with the most

potent of the aryloxypiperidine series described (Table 5).

Methyl substitution at the benzylic position (40) was not tolera-

ted and was not further explored. Substitution of the aromatic

ring revealed subtle diﬀerences for the small ﬂuorine substituent

(41ꢀ43) and more pronounced preference for the para position

with the bulkier triﬂuoromethyl derivatives (44ꢀ46). Broader

exploration of para-substitution indicated that hydrophobic

substitution was preferred but with a limit to the amount of

steric bulk that this region of chemical space would accommo-

date. For example, p-Me and p-i-Pr substitution aﬀorded sub-

micromolar compounds 47 and 48, respectively, but there was a

dramatic loss in potency with t-Bu substitution (49). Polar

substituents with hydrogen bond donors, such as primary

carboxamide 50, also resulted in a substantial loss of potency.

Electron rich substitutents, such as p-OMe (51), were not tole-

rated. Alternatively, substitution of the para position with a

triﬂuoromethoxy group (52) aﬀorded a potent analogue (0.5 μM).

Although alkyl substituted derivatives 47 and 48 aﬀorded

submicromolar potency, they were not further pursued because

of relatively poor selectivity over the hERG channel (IC₅₀of

1.93 and 0.44 μM, respectively) compared to 46 and 52,

(IC₅₀> 10 μM). The corresponding aryloxy derivatives contain-

ing p-triﬂuoromethyl (53) and triﬂuoromethoxy (54) groups

also exhibited submicromolar potency (0.8 and 0.3 μM,

respectively) with good microsomal stability in rat microsomes

(HLM/RLM CL_int= 157/38 and 110/33 (μL/min)/mg,

respectively). Combining the optimized piperidine aryl ether

fragment with the previously identiﬁed pyrimidine core (12)

yielded 55, which had submicromolar potency (0.9 μM)

but higher CL_intin RLM (HLM/RLM CL = 117/193 versus

91/46 ((μL/min)/mg) for 52). Thus, compounds that imparted

the greatest balance of potency and microsomal stability were

further evaluated in terms of selectivity and pharmacokinetic

properties.

In order to determine the best candidate for evaluation in pain

models, a more detailed selectivity and PKDM analysis of 46 and

52ꢀ54 was conducted (Table 6). Comparison of aryloxy ether

leads (53, 54) with benzyloxy ether analogues (46, 52) revealed

that the benzyloxy ether subseries demonstrated superior selectivity

over Nav1.5 and the hERG potassium channel and were there-

fore chosen for evaluation in rat iv and po PK studies. Gratify-

ingly, the low CL_intin RLM of 46 and 52 translated into low

clearance (0.48 and 0.42 L/(h kg), respectively). Furthermore,

3

compounds 46 and 52 were found to possess moderate and good

oral bioavailability (27% and 67%, respectively). Compound 52

was selected for further proﬁling and evaluation in the rat

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Table 3. Potency, PK, and Solubility Properties of Selected Derivatives^a

^aRat PK studies were conducted with male SpragueꢀDawley rats as follows: iv dosing, 0.5 mg/kg in 0.5 mL/kg of 100% DMSO; oral administration,

2 mg/kg in 10 mL/kg of vehicle (30% hydroxypropyl-β-cyclodextrin, pH 2.2 adjusted with methanesulfonic acid for 35 or 1% Tween 80, 2%

hydroxypropyl methylcellulose, pH 2.2 adjusted with HCl for 36). Data were obtained from MDR1-LLC-PK1 cells, pig kidney cells expressing human

MDR1, at 5 μM in the presence of 0.1% BSA, and permeability was estimated by averaging apparent permeability values in the apical to basolateral and

basolateral to apical directions.

formalin model of persistent pain based on its balance of potency,

selectivity, and oral exposure.

state-dependent inhibition of cloned channels and its potent

inhibition of Nav1.7 and Nav1.3, 52 (at 1 μM) inhibited the fast-

inactivating TTX-sensitive sodium channels of sensory neurons

(90% inhibition) but only with the holding voltage set to produce

partial channel inactivation. Compound 52 weakly inhibited

(15% inhibition) the slow-inactivating TTX-resistant sodium

channels of sensory neurons.

In addition to its strong state dependence, 52 also displayed

some selectivity within the sodium ion channel family (Table 7).

Selectivity for Nav1.7 over the tetrodotoxin resistant Nav1.5 and

Nav1.8 subtypes was modest but clear, at 6.5-fold and 13-fold,

respectively, whereas selectivity over Nav1.3 and Nav1.4 was not

observed.^35ꢀ37A greater than 50-fold selectivity margin over the

hERG channel and a greater than 100-fold margin over the Kv1.5

channel was observed. Compound 52 exhibited weak or no acti-

vity against an extended panel of pain related targets (TRPM8,

TRPV3, TRPV4, P₂X₇), a GPCR panel of 13 receptors, and a

CEREP panel. In addition, when 52 was screened in an Ambit

kinase panel (400 kinases) at 1 μM, no kinase was inhibited by

greater than 75% and only 13 kinases were inhibited by more

than 50% (Table 7).

For a more accurate measure of activity, compound 52 was

evaluated using manual whole-cell patch-clamp electrophysiol-

ogy and was conﬁrmed as a potent and state-dependent blocker

of Nav1.7, with IC₅₀= 170 nM on channels that were 20%

inactivated and IC₅₀= 3.6 μM on fully noninactivated channels

(average, n = 2 trials). All of the more than 20 analogues tested

with this protocol showed strong state dependence. Application

of the modulated receptor model with single aﬃnities for resting

(K_R) and inactivated (K_I) channels^32ꢀ34gives an extrapolated

aﬃnity of K_I= 35 nM for binding of 52 to fully inactivated

channels and a ratio K_R/K_I= 103. That is, the compound is

approximately 100-fold more potent on inactivated than on

resting (noninactivated) channels. By comparison, mexiletine

measured with the same protocol had IC₅₀= 56 μM on partially

inactivated Nav1.7 and IC₅₀= 340 μM on noninactivated

Nav1.7, for an extrapolated K_I= 13 μM and ratio K_R/K_I= 26,

demonstrating weaker state dependence than 52. To verify the

suitability of 52 for testing in rat models of pain, 52 was tested on

sodium channels recorded from cell bodies of individual sensory

neurons dissociated from rat dorsal root ganglia (DRG) (see

Supporting Information, Figure S1). Consistent with its strongly

The formalin model measures spontaneous rather than

evoked pain and involves the sensitization of central neurons

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Table 4. Comparison of Rat Liver Microsomal Clearance and Potency between des-Methyl and Methylated LHS Derivatives

ARTICLE

in response to a peripheral insult. This situation is perhaps

somewhat analogous to the clinical conditions of erythromelalgia

and paroxysmal extreme pain disorder, which are considered to

be spontaneous pain disorders triggered by activation of Nav1.7,

likely in peripheral neurons.⁵The formalin model is also appeal-

ing since its automated end point removes operator bias from the

quantitation of animal pain.^38,39

eﬃcacy at approximately 3 μM (20 mg/kg dose). The potency

of 52 incubated with 5% human serum albumin (HSA) on

Nav1.7 (20% inactivated, PX) was 3.3 μM. The unbound

plasma concentration (0.014 μM) at the minimum eﬃcacious

dose (3 mg/kg) was approximately 10- to 15-fold below the

measured IC₅₀on partially (∼20%) inactivated Nav1.7 channels

(0.17 μM). Human and rat Nav1.7 amino acid sequences are 96%

homologous and 93% identical; no pharmacological diﬀerences

between human and rat are known for existing sodium channel

blockers.

Compound 52 showed dose-dependent eﬃcacy in the for-

malin model, with statistically signiﬁcant analgesic eﬀects at

3 mg/kg, matched by dose-dependent increases in plasma con-

centration (Figure 2A, Supporting Information Figure S2). The

maximum eﬀect (20ꢀ30 mg/kg) was equivalent to that produced

by morphine. To verify that this apparent analgesic eﬃcacy was not

due to sedation or other eﬀects causing a nonspeciﬁc reduction in

movement, the same doses of 52 were also tested in a rat open

ﬁeld movement assay (Figure 2B, Supporting Information Figure

S2). Total plasma concentrations were comparable between the

open ﬁeld and the formalin experiment. The 3 and 10 mg/kg

doses of 52 were not signiﬁcantly diﬀerent from the vehicle

control. While the 20 mg/kg dose elicited a statistically signiﬁ-

cant 29% reduction in movements, the reduction exhibited was

not to the extent where conclusions drawn from the formalin test

would be deﬁnitively confounded. Typically a large reduction in

open ﬁeld activity, approximately 50% or more, needs to be

achieved before interpretation of a therapeutic eﬀect in the

formalin assay could be confounded.⁴⁰Once this threshold is

crossed, the rat has diﬃculty responding naturally to formalin

stimulus. The 30 mg/kg dose also produced a signiﬁcant reduc-

tion in open ﬁeld movement. Because of the 52% reduction in

movement produced at this dose, interpretation of the formalin

result is diﬃcult for reasons stated above. We conclude that

compound 52 gives analgesic eﬃcacy starting at plasma concen-

trations of approximately 0.5 μM (3 mg/kg dose), with full

Biochemically, sodium channels have six known binding sites

for neurotoxins and one deﬁned binding site for small molecule

inhibitors such as local anesthetics.^41,42The binding epitope for

local anesthetics, formed mostly by residues in the sixth trans-

membrane domain of the third and fourth pseudosubunits of the

sodium channel peptide sequence,⁴³has signiﬁcant overlap with

the binding site for batrachotoxin.^42,44ꢀ46Although batrachotox-

in binding is often used as a driver assay for the discovery and

characterization of sodium channel inhibitors,⁴⁷its binding epitope

is highly conserved among sodium channel isoforms, suggesting

that inhibitors acting at a diﬀerent site might be better candidates

for building an Nav1.7-selective molecule. To help understand

the observed subtype selectivity of 52, displacement assays were

performed with tritiated batrachotoxin (³H-BTX) and with a

tritiated triazine lead compound (³H-36) (Table 8, Supporting

Information Figure S3). Both ³H-BTX and ³H-36 showed

saturable speciﬁc binding to membranes from Nav1.7-expressing

3

cells. Interestingly, 52 did not displace H-BTX (K_i> 10 μM)

binding to Nav1.7 but did displace ³H-36 with K_i= 0.4 μM, close

to its IC₅₀on partially inactivated Nav1.7. By contrast, as

expected, the local anesthetic tetracaine inhibited ³H-BTX bind-

ing with K_i= 0.5 μM, again very close to its IC₅₀= 0.3 μM on

∼20% inactivated Nav1.7. Tetracaine had little eﬀect on ³H-36

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Table 5. SAR: Variations to the RHS Piperidine

binding (K_i= 12 μM), again consistent with separate binding

sites for triazines and for batrachotoxin/local anesthetics. Results

show that 52 (and likely other compounds within the triazine

chemical series) do not bind at the consensus local anesthetic/

batrachotoxin site of Nav1.7 and suggest that inhibitors discussed

herein recognize an undescribed epitope for small molecules.

Since compound 52 has some selectivity for diﬀerent sodium

channels, this binding site may deﬁne an epitope to which

diﬀerent sodium channel isoforms contribute diﬀerent amino

acid residues.

potent but highly state-dependent inhibitor of Nav1.7 with poor

PK properties. A broad structureꢀactivity relationship evalua-

tion with emphasis on optimization of potency and PK produced

compound 52, a more potent and strongly state dependent

inhibitor of Nav1.7 that demonstrated dose and exposure depen-

dent eﬃcacy in the formalin model of pain. Compound 52 had

selectivity over many ion channels and GPCRs, including some

selectivity within the sodium channel family, likely acting via a

novel site. Accordingly, the triazine series described here, includ-

ing compound 52, may be valuable for the further investigation of

the in vivo physiological roles of tetrodotoxin-sensitive sodium

channels in pain pathways.

In conclusion, a high-throughput screening campaign using a

functional electrophysiology platform identiﬁed 24 as a modestly

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Table 6. Selectivity and Oral PK Comparisons of 53, 54, 46, and 52^a

^aRat PK studies were conducted as follows: iv dosing, 0.5 mg/kg using 100% DMSO; with oral dosing, 2 mg/kg dosing using a formulation of 1%

hydroxypropyl methylcellulose (HPMC), 5% Tween 80, 94% water.

’ EXPERIMENTAL SECTION

inactivation produced by a given voltage. Current was evoked with a

20 ms test pulse to ꢀ20 or ꢀ10 mV, and current amplitudes were

corrected for leak by subtracting the current at the end of the test pulse

(leak current) from the peak current during the test pulse (sodium

channel current plus leak current). IC₅₀values for fully noninactivated

(resting) channels were determined with the holding voltage set to

either ꢀ140 or ꢀ120 mV (reported in brackets). For automated planar

patch-clamp recordings, cells were used in suspension as per manufac-

turer’s protocols. The voltage dependence of inactivation was measured

for each cell individually, and the holding voltage was set to produce 20%

fractional inactivation before application of each concentration of test

compound. Time courses of current were corrected for rundown

manually with DataXpress software. To record from individual rat dorsal

root ganglion neurons, the dorsal root ganglia were dissected from rats

aged 3ꢀ10 days and the ganglia enzymatically digested with collagenase

and Dispase followed by papain as described.⁴⁹Individual neurons were

released via gentle trituration and plated into a 35 mm dish and used on

the same day or the first day after dissection. Currents were evoked by

20 ms depolarizations to a voltage at or near the peak of the currentꢀ

voltage curve: approximately ꢀ20 mV for TTX-sensitive sodium

currents and þ10 mV for TTX-resistant sodium currents. At least four

different concentrations of test compound at half log units were applied

individually, with washout, recovery of current, and resetting of holding

voltage between each individual concentration. Percent inhibition as a

function of compound concentration was pooled from at least n = 10

different cells, with two to three data points per concentration, to

produce a single IC₅₀curve. A benchmark aminotriazine run as positive

Electrophysiology. Sodium currents were recorded with the

whole-cell configuration of the patch-clamp from Nav1.7 expressed in

HEK293 cells using either conventional operator recording (manual

electrophysiology as described)⁴⁸or with the PatchXpress planar patch

clamp automated electrophysiology system (Molecular Devices, Inc.).

For manual recordings, cells were cultured under standard conditions,

split, and plated into 35 mm dishes either 1 day before or on the same

day of recording, and culture medium was exchanged for external

solution containing (in mM) NaCl 140, KCl 2, CaCl₂2, MgCl₂1.1,

HEPES 10, glucose 11, pH 7.4 (NaOH). Internal solution contained

(in mM) CsCl 62.5, CsF 75, MgCl₂2.5, EGTA 5, HEPES 10, pH 7.2

(CsOH). Following establishment of the whole-cell patch-clamp con-

figuration, cells were lifted off the bottom of the dish with the patch pipet

and positioned directly in front of a microarray of glass tubes (each tube

internal diameter, ∼1 mm) with each tube containing continuously

flowing control or test solution. A full currentꢀvoltage relation was

recorded from each cell, and cells with poor space clamp as indicated by

discontinuous IꢀV relation or by prolonged or nonexponential current

kinetics were discarded. For each sodium channel, IC₅₀values for test

compounds were measured with a holding voltage set to produce

approximately 20% steady-state fractional inactivation, and this voltage

was reset as needed after application and washoff of each individual

compound concentration to maintain fractional inactivation at 20%. For

measurement of state-dependent pharmacology, the relevant parameter

is not the absolute voltage to which the cell is clamped, but the fractional

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Table 7. In Vitro Selectivity Proﬁle of 52

receptor

IC₅₀(μM)

hNav1.7^a

hNav1.5^a

hNav1.3^a

hNav1.4^a

rNav1.8^a

0.17 (3.6)

1.1 (>10)

0.3 (>10)

0.4 (>10)

2.2

hERG^b

>10

hTRPV3, hTRPV4^b

hTRPM8^b

Kv1.5^b

>10

>20

>33.3

b

rP₂X₇

>10

Cerep (at 10 μM)^c

A3 (h), TP (TXA2/PGH2) (h),

σ (nonselective), Na^þ(site 2)

(85% inhibition)

GPCR panel (13)

kinase (Ambit)^d

>50

0, 13, 387

^aIC₅₀numbers for sodium channels displayed in this table were taken

with manual electrophysiology on ∼20% inactivated channels. The

value in parentheses is the IC₅₀recorded on fully noninactivated

channels determined with a very negative holding potential, usually

ꢀ140 mV. ^bIC₅₀values for hERG and Kv1.5 were recorded with

electrophysiology; values for TRP and P2X receptors were recorded

with ﬂuorescence-based live-cell functional assays. ^cDisplayed >70%

inhibition of control speciﬁc binding at indicated receptor. Site 2 Na^þ

was measured from rat cortex, which contains a mix of sodium channel

isoforms and likely minimal Nav1.7, possibly accounting for the lower

aﬃnity seen for binding to hNav1.7 directly (Figure 2). ^dNumber of

kinases exhibiting % inhibition in the presence of 1 μM 52: >50%,

25ꢀ50%, and <25%, respectively.

Figure 2. (A) Time course of formalin-induced ﬂinches in animal

cohorts administered vehicle, morphine sulfate (subcutaneous dosing,

2 mg/kg), or 3, 10, 20, or 30 mg/kg compound 52: (left y-axis) total

ﬂinches in phase 2a as a function of dose; (right y-axis) mean plasma

concentrations in the formalin experiment for 3, 10, 20, 30 mg/kg doses

were 0.5, 1.4, 3.2, 4.4 μM. (B) (left y-axis) time course of movement

following administration of either vehicle or compound; (right y-axis)

mean plasma concentrations in the open-ﬁeld experiment for 3, 10, 20,

30 mg/kg doses were 0.5, 1.4, 2.9, 5.4 μM. /, p < 0.05, ///, p < 0.001

versus vehicle group (one-way ANOVA followed by Dunnett’s test).

control produced mean IC₅₀= 1.4 μM and standard deviation of 0.6 μM

from n = 25 individually calculated IC₅₀curves.

hERG (human ether-a-go-go-related gene) currents were recorded

with the whole-cell conﬁguration of the patch clamp from hERG

expressed in HEK293 cells (Millipore) using the PatchXpress planar

patch-clamp automated electrophysiology system (Molecular Devices,

Inc.) per the manufacturer’s protocol. The external recording solution

contained (in mM) NaCl (135), KCl (4), MgCl₂6H₂O (1), CaCl₂

(1.8), HEPES (10), and glucose (10). pH was adjusted to 7.4 with

NaOH, and the ﬁnal osmolarity was set at 310 mOsm. The internal

3

of the methods of Brown.^{50 3}H-BTX binding was assayed in 96-well

plates with 50 μg of membrane in each well, in binding buffer containing

(in mM) choline chloride 130, HEPES 50, glucose 5.5, MgSO₄0.8, KCl

5.4, plus 0.1% BSA (Sigma) and 1 protease inhibitor cocktail tablet

(Roche) per 50 mL of solution. Reactions were incubated for 1 h at

200 rpm shaking speed, and the product was filtered over 0.5% poly-

ethylenimine-treated GF/C filter plates (Perkin-Elmer), washed six times,

and dried. Counts were assayed with a MicroBeta imager (Perkin-Elmer),

and binding was expressed as counts per minute (CPM). Nonspecific

binding was determined by co-incubation with 100 μM veratridine

(³H-BTX binding) or of excess of cold compound 36. B_maxand K_dwere

calculated from the Langmuir equation. K_ivalues for individual com-

pounds were determined from experimentally determined IC₅₀via K_i=

IC₅₀/(1 þ (radioligand concentration)/K_d). For competition binding

experiments, radioligands were used at concentrations near K_d.

In Vivo Pharmacology. Formalin and open-field testing were

carried out as previously described.⁴⁸In brief, 50 μL of 2.5% formalin

diluted in saline was injected intraplantar, and flinching behaviors were

quantitated for the 40 min following injection using an automated

nociception analyzer from the University of California, San Diego.³⁸

Formalin evoked the characteristic biphasic response in which phase 2a

flinches (10ꢀ40 min after formalin injection) represent persistent pain

recording solution contained (in mM) KCl (70), KF (60), MgCl₂

6

3

H₂O (2), HEPES (10), EGTA (10), and MgATP (5). pH was adjusted

to 7.4 with KOH, and the ﬁnal osmolarity was set at 280 mOsm.

Cells were voltage clamped at ꢀ80 mV holding potential, and the

hERG current was activated by a depolarizing step ﬁrst to ꢀ50 mV for

500 ms to serve as a baseline for the outward tail current, then to þ30 mV

for 2 s to activate the channels, and ﬁnally back to ꢀ50 mV for 2 s to

remove the inactivation and record the deactivating outward tail current.

All test articles in solid format were dissolved in 100% dimethylsulf-

oxide (DMSO, Sigma) to a ﬁnal stock concentration of 10 mM. Serial

dilutions were prepared in DMSO, and 3 μL aliquots were subsequently

transferred to 900 mL of external recording solution in a 96 well plate

containing glass vials to minimize compound lost because of nonspeciﬁc

binding. The ﬁnal DMSO concentration in the external recording

solution was 0.3%. The compound plate was then sonicated for 15 min

before each experiment to ensure adequate mixing of the test article in

external recording solution.

Binding. Membranes were prepared from 293 cells stably expressing

Nav1.7, and binding of tritiated batrachotoxin (Perkin-Elmer) and of

radiolabeled triazine (compound ³H-36) was tested with modifications

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Table 8. Epitope Binding Aﬃnities for 52 and Tetracaine at BTx and ³H-36 Binding Sites

associated with central sensitization. Test compound was administered po

120 min prior to formalin injection to n = 8 animals per dose in vehicle of

30% hydroxypropyl-β-cyclodextrin, pH set to 1.5 with methane sulfonic

acid. Positive control morphine sulfate was administered sc 30 min prior to

formalin injection to n = 8 animals in a vehicle of saline. Movement of naive

(non-formalin-treated) animals in an open-field box was assayed with a

photobeam activity system (San Diego Instruments) and reported as

number of beam breaks binned in 5 min intervals and as total beam breaks

over the entire test session. Test compounds (n = 8 animals per dose) were

administered such that movement recording began at the same time after

compound administration as did the beginning of the formalin model.

Statisticalsignificance was asfollows: /, p< 0.05, ///, p<0.001byone-way

ANOVA and Dunnett’s post hoc test.

compounds was greater than 95% unless otherwise noted and was

measured using Agilent 1100 series high performance liquid chroma-

tography (HPLC) systems with UV detection at 254 nm (system A,

Agilent Zorbax Eclipse XDB-C8 4.6 mm ꢁ 150 mm, 5 μm, 5ꢀ100%

CH₃CN in H₂O with 0.1% TFA for 15 min at 1.5 mL/min; system B,

Waters Xterra 4.6 mm ꢁ 150 mm, 3.5 μm, 5ꢀ95% CH₃CN in H₂O with

0.1% TFA for 15 min at 1.0 mL/min). Exact mass confirmation was

performed on an Agilent 1100 series high performance liquid chroma-

tography (HPLC) system (Santa Clara, CA, U.S.) by flow injection

analysis, eluting with a binary solvent system A and B (A, water with

0.1% FA; B, ACN with 0.1% FA) under isocratic conditions (50% A/

50% B) at 0.2 mL/min with MS detection by an Agilent G1969A time of

1

flight (TOF) mass spectrometer (Santa Clara, CA, U.S.). H NMR

Chemistry. General. Unless otherwise noted, all materials were

obtained from commercial suppliers and used without further purifica-

tion. Dry organic solvents (CH₂Cl₂, CH₃CN, DMF, etc.) were pur-

chased from Aldrich packaged under nitrogen in Sure/Seal bottles.

Reactions were monitored using Agilent 1100 series LCꢀMS with UV

detection at 254 nm and a low resonance electrospray mode (ESI).

Medium pressure liquid chromatography (MPLC) was performed on a

CombiFlash Companion (Teledyne Isco) with RediSep normal-phase

silica gel (35ꢀ60 μm) columns or Biotage Isolera using SNAP columns

normal phase silica and UV detection at 254 nm. Preparative reversed-

phase HPLC was performed on a Gilson (215 liquid handler), YMC-

Pack Pro C18, 150 mm ꢁ 30 mm i.d. column, eluting with a binary

solvent system A and B using a gradient elution (A, H₂O with 0.1% TFA;

B, CH₃CN with 0.1% TFA) with UV detection at 254 nm. The analytical

SFC chromatograph was a SFC method development station sold by

Thar (Pittsburgh, PA, U.S.) equipped with a Waters ZQ mass spectro-

meter (Milford, MA, U.S.). The preparative SFC chromatograph was a

SFC Prep 80 from Thar (Pittsburgh, PA, U.S.). Purity for final

spectra were recorded on a Bruker AV-400 (400 MHz) spectrometer at

ambient temperature or on a Varian 400 MHz spectrometer. Chemical

shifts are reported in ppm from the solvent resonance (DMSO-d₆2.50

ppm). Data are reported as follows: chemical shift, multiplicity (s =

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

coupling constants, and number of protons.

N-(3-(4-Chloro-1,3,5-triazin-2-ylamino)phenyl)acetamide

(3). To a brown solution of N-(3-aminophenyl)acetamide (8.19 g, 54.5

mmol) in 1,2-dimethoxyethane (81 mL) was added diisopropylethyla-

mine (23.7 mL, 136 mmol), and the mixture was cooled in an iceꢀwater

bath. A suspension of 2,4-dichloro-1,3,5-triazine (9.0 g, 60.0 mmol) in

1,2-dimethoxyethane (195 mL) was cooled in an iceꢀwater bath, then

added to the cooled N-(3-aminophenyl)acetamide solution. The solu-

tion was allowed to stir and warm on its own to room temperature for

16 h. To the pale brown solution was added ethyl acetate and 1 M HCl

(400 mL). The aqueous phase was extracted with ethyl acetate (300 mL,

2ꢁ). The combined organics were dried with Na₂SO₄, filtered, and dried

under reduced pressure. Upon standing a solid formed in the aqueous

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phase, which was collected by filtration. The combined solids were

triturated with CH₂Cl₂to remove any yellow impurities, providing the

title compound (13.18 g, 91.7% yield) as a light yellow solid. LCꢀMS

(ESI) m/z: 264.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 10.74 (s,

1H), 10.04 (s, 1H), 8.62 (s, 1H), 7.87 (br s, 1H), 7.39 (br. s, 1H),

7.34ꢀ7.22 (m, 2H), 2.04 (s, 3H).

dissolved in EtOAc, transferred to a separatory funnel, and washed with

saturated aqueous NaHCO₃. The organic phase was washed with brine.

The combined organic washes were dried with Na₂SO₄, ﬁltered, and

dried under reduced pressure. The crude residue obtained was puriﬁed

by silica gel chromatography (0ꢀ100% CH₂Cl₂/CH₃OH (90:10) in

CH₂Cl₂), providing the title compound (176 mg, 80%) as a white solid.

LCꢀMS (ESI) m/z: 360.0 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ

9.79 (s, 1H), 9.01 (s, 1H), 8.11 (s, 1H), 7.98 (d, J = 6.06 Hz, 1H), 7.33

(d, J = 7.83 Hz, 1H), 7.28ꢀ7.17 (m, 4H), 7.14 (t, J = 8.00 Hz, 1H), 7.07

(d, J = 8.00 Hz, 1H), 6.30 (d, J = 6.06 Hz, 1H), 4.77 (s, 2H), 3.83 (t, J =

5.33 Hz, 2H), 2.90 (t, J = 5.87 Hz, 2H), 2.05 (s, 3H). HRMS calcd for

C₂₁H₂₁N₅O (M þ H)^þ360.1819, found 360.1823.

N-(3-(4-Chloro-1,3,5-triazin-2-ylamino)-2-methylphenyl)-

acetamide (22). A solution of 2,4-dichloro-1,3,5-triazine (5.00 g, 33.3

mmol) in N,N-dimethylformamide (44.5 mL) and N,N-diisopropy-

lethylamine (6.38 mL, 36.7 mmol) was cooled in an iceꢀwater bath,

and N-(3-amino-2-methylphenyl)acetamide (5.47 g, 33.3 mmol, 71.7%

yield) was added. The mixture was allowed to slowly warm to room

temperature and was stirred for a total of 18 h. To the resulting dark

orange solution was added water (∼200 mL) and the turbid mixture

cooled in the refrigerator overnight leading to the formation of a yellow

precipitate which was collected via vacuum filtration and the solid

washed with minimal ethyl acetate and excess water. The solid was

dried under reduced pressure to provide the title compound (6.64 g,

72%) as a yellow solid. LCꢀMS (ESI) m/z: 278.1 (M þ 1). ¹H NMR

(400 MHz, DMSO-d₆) δ 10.27 (s, 1H), 9.39 (br s, 1H), 8.38ꢀ8.60 (m,

1H), 7.31 (d, J = 6.65 Hz, 1H), 7.19 (t, J = 8.00 Hz, 1H), 7.12 (d, J = 8.00

Hz, 1H), 2.06 (s, 3H), 2.03 (s, 4H).

N-(3-(6-(3,4-Dihydroisoquinolin-2(1H)-yl)pyrimidin-4-yla-

mino)phenyl)acetamide (12). To a flask charged with 4,6-dichlor-

opyrimidine (250 mg, 1.68 mmol) were added 2-propanol (6.7 mL) and

N,N-diisopropylethylamine (308 μL, 1.76 mmol). The solution was

cooled in an iceꢀwater bath prior to the addition of 1,2,3,4-tetrahy-

droisoquinoline (224 μL, 1.68 mmol). The mixture was stirred and

allowed to slowly warm on its own to room temperature, leading to the

formation of a white precipitate after 1 h. The solid was collected via

vacuum filtration and the solid washed with excess 2-propanol, then

dried under reduced pressure, providing a white solid 2-(6-chloropyr-

imidin-4-yl)-1,2,3,4-tetrahydroisoquinoline (11) (340 mg, 1.384 mmol,

82% yield). LCꢀMS (ESI) m/z: 246.0 (M þ 1).

N-(3-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)pyrimidin-4-yla-

mino)phenyl)acetamide (7). To a flask charged with 2,4-dichlor-

opyrimidine (1.00 g, 6.7 mmol) in 15 mL of 2-propanol at 0 ꢀC was

added diisopropylethylamine (2.33 mL, 13.4 mmol) followed by N-(3-

aminophenyl)acetamide (1.01 g, 6.7 mmol). The resulting mixture was

stirred at 0 ꢀC for 15 min, then at ambient for 21 h. The resulting mixture

was dried under reduced pressure and purified by silica gel chromatog-

raphy (0ꢀ100% ethyl acetate in hexanes), providing (6) N-(3-(2-

chloropyrimidin-4-ylamino)phenyl)acetamide (554 mg, 31%). LCꢀMS

(ESI) m/z: 263.0 (M þ 1).

To a ﬂask charged with 2-(6-chloropyrimidin-4-yl)-1,2,3,4-tetrahy-

droisoquinoline (150 mg, 0.610 mmol) was added 2-propanol (4.1 mL)

followed by 3⁰-aminoacetanilide (110 mg, 0.733 mmol) and triﬂuor-

oacetic acid (118 μL, 1.53 mmol). The vessel was sealed and heated

overnight at 95 ꢀC, providing a light brown suspension. The mixture was

cooled to room temperature, then in a ꢀ20 ꢀC freezer for 1 h. The result-

ing solid was collected via vacuum ﬁltration and washed with excess

2-propanol. The ﬁltrate was concentrated under reduced pressure and

the oil obtained puriﬁed by silica gel chromatography (0ꢀ100%

CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in CH₂Cl₂), providing the title

compound (75 mg, 34.2% yield) as a white solid. LCꢀMS (ESI) m/z:

360.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.87 (s, 1H), 9.07 (s,

1H), 8.22 (d, J = 0.59 Hz, 1H), 7.88ꢀ7.85 (m, 1H), 7.28 (td, J = 1.82,

7.52 Hz, 1H), 7.26ꢀ7.11 (m, 6H), 6.08 (d, J = 0.59 Hz, 1H), 4.67 (s,

2H), 3.76 (t, J = 5.92 Hz, 2H), 2.89 (t, J = 5.87 Hz, 2H), 2.04 (s, 3H).

HRMS calcd for C₂₁H₂₁N₅O (M þ H)^þ360.1819, found 360.1823.

N-(3-((4-(3,4-Dihydroisoquinolin-2(1H)-yl)-1,3,5-triazin-2-

yl)(methyl)amino)phenyl)acetamide (15). To a flask charged

with 2,4-dichloro-1,3,5-triazine (0.500 g, 3.33 mmol) was added N,N-

dimethylformamide (13.3 mL) and N,N-diisopropylethylamine (1.17 mL,

6.67 mmol), respectively. The resulting yellow solution was cooled in an

iceꢀwater bath prior to the addition of 3-bromo-N-methylaniline

(0.425 mL, 3.33 mmol). The solution was allowed to stir and warm

on its own to room temperature over 3.5 h, leading to clean conversion

to desired triazine monochloride species. To the mixture was added

1,2,3,4-tetrahydroisoquinoline (0.444 mL, 3.33 mmol), and stirring at

room temperature continued overnight. The orange solution was dried

under reduced pressure and the crude material purified by silica gel

chromatography (0ꢀ100% CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in

CH₂Cl₂), providing a mixture of product and a major aliphatic impurity.

The orange oil was dissolved in DCM and diethyl ether was added

leading to the formation of an orange precipitate which was collected via

vacuum filtration and washed with diethyl ether. The filtrate was dried

under reduced pressure, providing product as an orange oil, N-(3-

bromophenyl)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-N-methyl-1,3,5-

triazin-2-amine (14) (336 mg, 0.848 mmol, 25.4%). LCꢀMS (ESI)

m/z: 396.0/398.0 (M, M þ 2), 2.329 min.

To a ﬂask charged with N-(3-(2-chloropyrimidin-4-ylamino)phenyl)-

acetamide (150.0 mg, 571 μmol) and 1,2,3,4-tetrahydroisoquinoline

(79.7 μL, 628 μmol) were added 2-propanol (3 mL) and triﬂuoroacetic

acid (132 μL, 1.71 mmol). The resulting mixture was heated at 90 ꢀC for

23 h. The resulting mixture was concentrated, dissolved in EtOAc,

transferred to a separatory funnel, and washed with saturated aqueous

NaHCO₃. The organic phase was washed with brine. The combined

aqueous washes were extracted with ethyl acetate. The combined organic

washes were dried with Na₂SO₄, ﬁltered, and dried under reduced pres-

sure. The crude residue obtained was puriﬁed by silica gel chromatography

(0ꢀ100% CH₂Cl₂/CH₃OH (90:10) in CH₂Cl₂), providing the title

compound (103 mg, 50%) as a white solid. LCꢀMS (ESI) m/z: 360.0

(M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.90 (s, 1H), 9.27 (s, 1H),

8.17 (br s, 1H), 7.95 (d, J = 5.67 Hz, 1H), 7.39 (d, J = 7.83 Hz, 1H),

7.28ꢀ7.11 (m, 5H), 7.00 (d, J = 7.92 Hz, 1H), 6.08 (d, J = 5.67 Hz, 1H),

4.86 (s, 2H), 3.97 (t, J = 5.87 Hz, 2H), 2.85 (t, J = 5.67 Hz, 2H), 2.07 (s,

3H). HRMS calcd for C₂₁H₂₁N₅O (M þ H)^þ360.1819, found 360.182.

N-(3-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)pyrimidin-2-yla-

mino)phenyl)acetamide (9). To a flask charged with 2,4-dichlor-

opyrimidine (1.00 g, 6.7 mmol) in 15 mL of 2-propanol at 0 ꢀC was

added diisopropylethylamine (2.33 mL, 13.4 mmol) followed by 1,2,3,4-

tetrahydroisoquinoline (0.894 g, 6.7 mmol). The resulting mixture was

stirred at 0 ꢀC for 15 min, then at ambient for 24 h. The resulting mixture

was dried under reduced pressure and purified by silica gel chromatogra-

phy (0ꢀ60% ethyl acetate in hexanes), providing (8) 2-(2-chloropyri-

midin-4-yl)-1,2,3,4-tetrahydroisoquinoline (1.45 g, 88%). LCꢀMS (ESI)

m/z: 246.0 (M þ 1).

To a ﬂask charged with 2-(2-chloropyrimidin-4-yl)-1,2,3,4-tetrahy-

droisoquinoline (150.0 mg, 610 μmol) and N-(3-aminophenyl)-

acetamide (101 mg, 672 μmol) were added 2-propanol (3 mL) and

triﬂuoroacetic acid (141 μL, 1.83 mmol). The resulting mixture was

heated at 90 ꢀC for 23 h. The resulting mixture was concentrated,

To a vial charged with N-(3-bromophenyl)-4-(3,4-dihydroisoquino-

lin-2(1H)-yl)-N-methyl-1,3,5-triazin-2-amine (192 mg, 0.485 mmol)

were added acetamide (172 mg, 2.91 mmol), xantphos (56.1 mg,

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0.097 mmol), cesium carbonate (631 mg, 1.94 mmol), and pd₂dba₃

(44.4 mg, 0.048 mmol). The vessel was placed under argon prior to the

addition of dry 1,4-dioxane (2.42 mL). The vessel was sealed and heated

to 110 ꢀC for 16 h. To the mixture was added water, and the material was

transferred to a separatory funnel and extracted with CH₂Cl₂(2ꢁ). The

combined organics were dried with Na₂SO₄, ﬁltered, and dried under

reduced pressure. The crude material was puriﬁed by silica gel chroma-

tography (0ꢀ100% CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in CH₂Cl₂),

providing the title compound (64 mg, 0.171 mmol, 35.3%) as a yellow

solid. LCꢀMS (ESI) m/z: 375.2 (M þ 1). ¹H NMR (400 MHz, DMSO-

d₆) δ 9.97 (s, 1H), 8.15 (s, 1H), 7.60 (t, J = 1.86 Hz, 1H), 7.43 (d, J =

8.22 Hz, 1H), 7.30 (t, J = 8.07 Hz, 1H), 7.22ꢀ7.15 (m, 4H), 7.00 (d, J =

7.73 Hz, 1H), 4.91ꢀ4.73 (m, 2H), 3.99ꢀ3.80 (m, 2H), 3.42 (s, 3H),

2.84 (t, J = 5.92 Hz, 2H), 2.04 (s, 3H). HRMS calcd for C₂₁H₂₂N₆O

(M þ H)^þ375.1928, found 375.1931.

J = 8.41 Hz, 1H), 8.31 (s, 1H), 7.97ꢀ7.74 (m, 1H), 7.66ꢀ7.52 (m, 1H),

7.38 (t, J = 8.02 Hz, 1H), 7.27ꢀ7.12 (m, 4H), 6.97 (d, J = 7.53 Hz, 1H),

4.88 (d, J = 11.54 Hz, 2H), 4.03ꢀ3.93 (m, 2H), 3.18 (t, J = 4.89 Hz, 3H),

2.89 (d, J = 2.84 Hz, 2H), 1.83 (br s, 3H). HRMS calcd for C₂₁H₂₂N₆O

(M þ H)^þ374.1928, found 374.1932.

N-(3-(4-Amino-1,3,5-triazin-2-ylamino)phenyl)acetamide

(26). The title compound was prepared using a method analogous to the

preparation of compound 27 using 2 M NH₃in MeOH (10 equiv). The

crude mixture was dried under reduced pressure and purified by silica gel

chromatography (0ꢀ100% CH₂Cl₂/CH₃OH (90:10) in CH₂Cl₂) to

provide the title compound as a white solid. Yield: 107 mg, 72%.

1

LCꢀMS (ESI) m/z: 245.2 (M þ 1). H NMR (400 MHz, DMSO-

d₆) δ 9.83 (s, 1H), 9.42 (s, 1H), 8.13 (s, 1H), 7.74 (s, 1H), 7.47 (d, J =

9.10 Hz, 1H), 7.29 (d, J = 8.22 Hz, 1H), 7.16 (t, J = 7.80 Hz, 1H),

7.09ꢀ6.81 (m, 2H), 2.03 (s, 3H). HRMS calcd for C₁₁H₁₂N₆O (M þ

H)^þ245.1145, found 245.115.

N-(3-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)-1,3,5-triazin-2-

yloxy)phenyl)acetamide (18). To a flask charged with 2,4-

dichloro-1,3,5-triazine (2.00 g, 13.3 mmol) in 10 mL of N,N-dimethyl-

formamide at 0 ꢀC was added N,N-diisopropylethylamine (2.32 mL,

13.3 mmol) followed by 1,2,3,4-tetrahydroisoquinoline (1.69 mL,

13.3 mmol). The resulting mixture was stirred at 0 ꢀC for 10 min, then

at ambient temperature for 2 h. Water was added to the mixture and the

resulting orange slurry stirred for 20 min. Ethyl acetate was added, and

the mixture was stirred for 10 min and transferred to separatory funnel.

The organic phase was washed with water and brine. The combined

aqueous washes were extracted with ethyl acetate (2ꢁ). The combined

organic washes were dried with Na₂SO₄, filtered, and dried under

reduced pressure. The crude residue was adsorbed onto silica gel and

purified by silica gel chromatography (0ꢀ40% ethyl acetate in hexanes),

providing (16) 2-(4-chloro-1,3,5-triazin-2-yl)-1,2,3,4-tetrahydroisoqui-

noline (1.76 g, 54%). LCꢀMS (ESI) m/z: 247.0 (M þ 1).

N-(3-(4-(Piperidin-1-yl)-1,3,5-triazin-2-ylamino)phenyl)-

acetamide (27). In a sealed tube charged with N-(3-(4-chloro-1,3,5-

triazin-2-ylamino)phenyl)acetamide (40 mg, 152 μmol) (3) was added

N,N-dimethylformamide (0.600 mL, 152 μmol), N,N-diisopropylethy-

lamine (0.055 mL, 319 μmol), and piperidine (0.018 mL, 182 μmol),

respectively. The tube was sealed and heated to 90 ꢀC. The resulting

brown solution was cooled to room temperature and water was added

providing a brown precipitate which was collected via vacuum filtration

and washed with water, then dried under high vacuum, providing the title

compound (39 mg, 82% yield) as a tan solid. LCꢀMS (ESI) m/z: 313.2

(M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H), 9.54 (br s, 1H),

8.20 (s, 1H), 8.05 (br s, 1H), 7.33ꢀ7.28 (m, 1H), 7.20ꢀ7.09 (m, 2H),

3.79ꢀ3.71 (m, 4H), 2.02 (s, 3H), 1.67ꢀ1.59 (m, 2H), 1.52 (br s, 4H).

HRMS calcd for C₁₆H₂₀N₆O (M þ H)^þ313.1771, found 313.1776.

N-(3-(4-(4-Methylpiperidin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (28). The title compound was prepared using a

method analogous to the preparation of 27. Crude solutions were

purified with preparative mass triggered HPLC using CH₃CN (with

0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the eluent, providing the

title compound. Yield: 52 mg, 60%. LCꢀMS (ESI) m/z: 327.2 (M þ 1).

¹H NMR (400 MHz, DMSO-d₆) δ 9.85 (s, 1H), 9.52 (s, 1H), 8.20 (s,

1H), 8.05 (br s, 1H), 7.30 (d, J = 7.34 Hz, 1H), 7.20ꢀ7.06 (m, 2H), 4.66

(br s, 2H), 2.91ꢀ2.82 (m, 2H), 2.02 (s, 3H), 1.74ꢀ1.57 (m, 3H),

1.11ꢀ0.97 (m, 2H), 0.92 (d, J = 6.26 Hz, 3H). HRMS calcd for

C₁₇H₂₂N₆O (M þ H)^þ327.1928, found 327.1933.

To a ﬂask charged with 3-acetamidophenol (91.9 mg, 608 μmol) in

3 mL N,N-dimethylformamide at ambient temperature was added

sodium hydride (60% in mineral oil) (23.3 mg, 608 μmol). The resulting

mixture was stirred at ambient temperature for 30 min, when 2-(4-

chloro-1,3,5-triazin-2-yl)-1,2,3,4-tetrahydroisoquinoline (150.0 mg, 608

μmol) was added in one portion. The resulting mixture was stirred at

ambient temperature for 17 h. The resulting mixture was quenched with

water, diluted with ethyl acetate, and transferred to a separatory funnel.

The organic layer was washed water and brine, dried with Na₂SO₄,

ﬁltered, and dried under reduced pressure. The crude residue was

puriﬁed by silica gel chromatography (0ꢀ90% ethyl acetate in hexanes),

providing the title compound (165 mg, 75%) as a white solid. LCꢀMS

(ESI) m/z: 362.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 10.06 (s,

1H), 8.43 (d, J = 4.21 Hz, 1H), 7.56 (br s, 1H), 7.41ꢀ7.30 (m, 2H),

7.27ꢀ7.14 (m, 4H), 6.88 (m, 1H), 4.91 (s, 1H), 4.76 (s, 1H), 4.06ꢀ3.97

(m, 1H), 3.82 (t, J = 5.82 Hz, 1H), 2.91ꢀ2.81 (m, 2H), 2.05 (s, 3H).

HRMS calcd for C₂₀H₁₉N₅O₂(M þ H)^þ362.1612, found 362.1618.

N-(3-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-1,3,5-triazin-2-

ylamino)phenyl)acetamide (24). See ref 25 for the procedure.

N-(3-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)-1,3,5-triazin-2-

ylamino)phenyl)-N-methylacetamide (25). To a flask charged

with 2,4-dichloro-1,3,5-triazine (91 mg, 0.609 mmol) was added N,N-

dimethylformamide (2.4 mL) followed by N,N-diisopropylethylamine

(319 μL, 1.827 mmol) and N-(3-aminophenyl)-N-methylacetamide

(100 mg, 0.609 mmol). The mixture was stirred at room temperature

for 30 min prior to the addition of 1,2,3,4-tetrahydroisoquinoline (77 μL,

0.609 mmol). The resulting solution was stirred for 16 h, then dried

under reduced pressure and purified by silica gel chromatography

(0ꢀ100% CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in CH₂Cl₂), leading

to isolation of the product along with trace impurities. The solid

obtained was triturated with MeOH/DCM (1:1) to provide the title

compound (12 mg, 0.032 mmol, 5.3% yield) as a white solid. LCꢀMS

(ESI) m/z: 375.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.82 (d,

N-(3-(4-(4-Methoxypiperidin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (29). The title compound was prepared using a

method analogous to the preparation of 27. Crude solutions were

purified with preparative mass triggered HPLC using CH₃CN (with

0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the eluent, providing the

title compound. Yield: 42 mg, 64%. LCꢀMS (ESI) m/z: 343.2 (M þ 1).

¹H NMR (400 MHz, DMSO-d₆) δ 9.84 (s, 1H), 9.54 (s, 1H), 8.21 (s,

1H), 8.07 (br s, 1H), 7.29 (d, J = 7.34 Hz, 1H), 7.20ꢀ7.09 (m, 2H),

4.21ꢀ4.09 (m, 2H), 3.50ꢀ3.38 (m, 3H), 3.28 (s, 3H), 2.03 (s, 3H),

1.93ꢀ1.78 (m, 2H), 1.48ꢀ1.33 (m, 2H). HRMS calcd for C₁₇H₂₂N₆O₂

(M þ H)^þ343.1877, found 343.1885.

1-(4-(3-Acetamidophenylamino)-1,3,5-triazin-2-yl)piperidine-

4-carboxamide (30). The title compound was prepared using a

method analogous to the preparation of 27. Crude solutions were

purified with preparative mass triggered HPLC using CH₃CN (with

0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the eluent, providing the

title compound. Yield: 53 mg, 60%. LCꢀMS (ESI) m/z: 356.0 (M þ 1).

¹H NMR (400 MHz, DMSO-d₆) δ 9.88 (s, 1H), 9.59 (s, 1H), 8.22 (s,

1H), 8.10 (br s, 1H), 7.33ꢀ7.25 (m, 2H), 7.17 (t, J = 7.97 Hz, 1H),

7.09ꢀ7.14 (m, 1H), 6.81 (s, 1H), 4.73ꢀ4.60 (m, 2H), 2.93 (dt, J = 2.64,

12.67 Hz, 2H), 2.39 (tt, J = 3.57, 11.44 Hz, 1H), 1.77 (d, J = 12.32 Hz,

2H), 1.53ꢀ1.38 (m, 2H). HRMS calcd for C₁₇H₂₁N₇O₂(M þ H)^þ

356.1829, found 356.1833.

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N-(3-(4-(4-Phenylpiperidin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (31). The title compound was prepared using a

method analogous to the preparation of compound 27. The crude mix-

ture was dried under reduced pressure and purified by silica gel chroma-

tography (0ꢀ100% CH₂Cl₂/CH₃OH (90:10) in CH₂Cl₂) to provide

the title compound as a white solid. Yield: 85 mg, 58%. LCꢀMS (ESI)

m/z: 389.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.84 (s, 1H),

9.56 (s, 1H), 8.23 (s, 1H), 8.10 (br s, 1H), 7.33ꢀ7.24 (m, 5H),

7.22ꢀ7.08 (m, 3H), 4.96ꢀ4.79 (m, 2H), 2.97 (dt, J = 2.45, 12.86 Hz,

2H), 2.84 (tt, J = 3.36, 12.09 Hz, 1H), 2.01 (s, 3H), 1.86 (d, J = 12.42 Hz,

2H), 1.65ꢀ1.52 (m, 2H). HRMS calcd for C₂₂H₂₄N₆O (M þ H)^þ

389.2084, found 389.2087.

N-(3-(4-(4-Benzylpiperidin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (32). The title compound was prepared using a

method analogous to the preparation of 27. Crude solutions were

purified with preparative mass triggered HPLC using CH₃CN (with

0.1% trifluoroacetic acid) in trifluoroacetic acid (0.1% aqueous) as the

eluent, providing the title compound as a trifluoroacetate salt. Yield: 47 mg,

40%. Purity: 92%. LCꢀMS (ESI) m/z: 403.2 (M þ 1). ¹H NMR (400

MHz, DMSO-d₆) δ 9.86 (s, 1H), 9.70 (s, 1H), 8.24 (s, 1H), 8.06 (s, 1H),

7.31ꢀ7.25 (m, 3H), 7.22ꢀ7.15 (m, 4H), 7.15ꢀ7.09 (m, 1H), 4.74ꢀ4.57

(m, 2H), 2.92ꢀ2.81 (m, 2H), 2.53 (d, J = 6.85 Hz, 2H), 2.01 (s, 3H),

1.88ꢀ1.78 (m, 1H), 1.66 (d, J = 12.03 Hz, 2H), 1.20ꢀ1.06 (m, 2H).

HRMS calcd for C₂₃H₂₆N₆O (M þ H)^þ403.2241, found 403.2244.

N-(3-(4-(4-Hydroxy-4-phenylpiperidin-1-yl)-1,3,5-triazin-

2-ylamino)phenyl)acetamide (33). The title compound was pre-

pared using a method analogous to the preparation of 27. Crude solu-

tions were purified with preparative mass triggered HPLC using CH₃CN

(with 0.1% trifluoroacetic acid) in trifluoroacetic acid (0.1% aqueous) as

the eluent, providing the title compound as a trifluoroacetate salt. Yield:

54 mg, 46%. LCꢀMS (ESI) m/z: 405.2 (M þ 1). ¹H NMR (400 MHz,

DMSO-d₆) δ 9.82 (s, 1H), 9.54 (s, 1H), 8.23 (s, 1H), 8.11 (br s, 1H),

7.50 (dd, J = 1.22, 8.36 Hz, 2H), 7.35ꢀ7.27 (m, 3H), 7.24ꢀ7.14 (m,

2H), 7.14ꢀ7.07 (m, 1H), 5.15 (s, 1H), 4.73ꢀ4.58 (m, 2H), 3.36ꢀ3.26

(m, 2H), 2.01 (s, 3H), 1.96ꢀ1.82 (m, 2H), 1.74ꢀ1.64 (m, 2H). HRMS

calcd for C₂₂H₂₄N₆O₂(M þ H)^þ405.2034, found 405.2033.

mixture was dried under reduced pressure and purified by silica gel

chromatography (0ꢀ100% ethyl acetate in hexanes) to provide the title

compound as a white solid. Yield: 954 mg, 62%. LCꢀMS (ESI) m/z:

419.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.85 (s, 1H), 9.56 (s,

1H), 8.22 (s, 1H), 8.08 (br s, 1H), 7.37ꢀ7.34 (m, 4H), 7.33ꢀ7.25 (m,

2H), 7.20ꢀ7.10 (m, 2H), 4.56 (s, 2H), 4.24ꢀ4.11 (m, 2H), 3.68 (tt, J =

3.85, 7.79 Hz, 1H), 3.52ꢀ3.42 (m, 2H), 2.03 (s, 3H), 1.96ꢀ1.86 (m,

2H), 1.58ꢀ1.45 (m, 2H). HRMS calcd for C₂₃H₂₆N₆O₂(M þ H)^þ

419.219, found 419.2194.

N-(3-(4-(4-(Benzyloxy)piperidin-1-yl)-1,3,5-triazin-2-ylamino)-

2-methylphenyl)acetamide (37). The title compound was pre-

pared using a method analogous to the preparation of 21 using precursor

22. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 110 mg, 70.6%. LCꢀMS

(ESI) m/z: 433.0 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s,

1H), 8.96 (s, 1H), 8.11 (s, 1H), 7.37ꢀ7.32 (m, 4H), 7.31ꢀ7.24 (m, 1H),

7.20ꢀ7.08 (m, 3H), 4.53 (s, 2H), 4.19ꢀ3.94 (m, 2H), 3.65 (tt, J = 3.72,

7.87 Hz, 1H), 3.48ꢀ3.32 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.91ꢀ1.82

(m, 2H), 1.52ꢀ1.41 (m, 2H). HRMS calcd for C₂₄H₂₈N₆O₂(M þ H)^þ

433.2347, found 433.2352.

N-(2-Methyl-3-(4-(4-phenylpiperidin-1-yl)-1,3,5-triazin-2-

ylamino)phenyl)acetamide (38). The title compound was pre-

pared using a method analogous to the preparation of 21 using precursor

22. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 17 mg, 17%. LCꢀMS (ESI)

m/z: 403.2 (M þ 1). ¹H NMR (500 MHz, DMSO-d₆) δ 9.35 (s, 1H),

9.02 (s, 1H), 8.13 (s, 1H), 7.28 (d, J = 8.55 Hz, 2H), 7.20ꢀ7.14 (m, 2H),

7.13ꢀ7.05 (m, 4H), 4.69ꢀ4.61 (m, 1H), 4.22ꢀ3.95 (m, 2H), 3.59ꢀ3.41

(m, 2H), 2.06ꢀ2.00 (m, 6H), 2.00ꢀ1.91 (m, 2H), 1.63ꢀ1.52 (m, 2H).

HRMS calcd for C₂₂H₂₆N₆ONa (M þ Na)^þ425.206, found 425.2066.

N-(2-Methyl-3-(4-(4-phenoxypiperidin-1-yl)-1,3,5-triazin-

2-ylamino)phenyl)acetamide (39). The title compound was pre-

pared using a method analogous to the preparation of 21 using precursor

22. Crude solutions were purified with preparative RP-HPLC using

CH₃CN (with 0.1% trifluoroacetic acid) in trifluoroacetic acid (0.1%

aqueous) as the eluent. Fractions containing the product were combined

and washed with saturated NaHCO₃. The product was extracted with

CH₂Cl₂. The organic phase was washed with brine, dried over MgSO₄,

filtered, and concentrated, providing the title compound. Yield: 53 mg,

35%. LCꢀMS (ESI) m/z: 419.0 (M þ 1). ¹H NMR (400 MHz, DMSO-

d₆) δ 9.35 (s, 1H), 9.02 (s, 1H), 8.13 (s, 1H), 7.32ꢀ7.23 (m, 2H),

7.21ꢀ7.14 (m, 2H), 7.13ꢀ7.07 (m, 1H), 6.98 (d, J = 7.82 Hz, 2H), 6.93

(t, J = 7.29 Hz, 1H), 4.68ꢀ4.58 (m, 1H), 4.23ꢀ3.95 (m, 2H), 3.63ꢀ3.38

(m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.99ꢀ1.90 (m, 2H), 1.64ꢀ1.52 (m,

2H). HRMS calcd for C₂₃H₂₆N₆O₂(M þ H)^þ419.219, found 419.2193.

N-(2-Methyl-3-(4-(4-(1-phenylethoxy)piperidin-1-yl)-1,3,

5-triazin-2-ylamino)phenyl)acetamide (40). To a solution of

tert-butyl 4-hydroxy-1-piperidinecarboxylate (369 μL, 2.5 mmol) in N,

N-dimethylformamide (8.3 mL, 2484 μmol) cooled in an iceꢀwater

bath was added sodium hydride (60% in mineral oil) (100 mg, 4.2 mmol).

The bath was removed and the suspension stirred at room temperature.

The suspension was stirred for 1 h at room temperature prior to the

addition of (1-bromoethyl)benzene (460 μL, 2.5 mmol) via syringe. The

resulting solution was stirred for 1 h at room temperature and heated at

90 ꢀC for 16 h. The mixture was cooled to room temperature, diluted

with water, and extracted with ethyl acetate. The organic layer was dried

with Na₂SO₄, filtered, and dried under reduced pressure, providing

crude tert-butyl 4-(1-phenylethoxy)piperidine-1-carboxylate. LCꢀMS

(ESI) m/z: 398.2 (M þ 23).

N-(3-(4-(4-Benzoylpiperazin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (34). The title compound was prepared using a

method analogous to the preparation of 27. The crude residue obtained

was purified by silica gel chromatography (0ꢀ100% CH₂Cl₂/CH₃OH/

NH₄OH (90:10:1) in CH₂Cl₂), providing the title compound as a white

solid. Yield: 42 mg, 15%. LCꢀMS (ESI) m/z: 418.2 (M þ 1). ¹H NMR

(400 MHz, DMSO-d₆) δ 9.84 (br s, 1H), 9.65 (s, 1H), 8.30ꢀ8.14 (m,

2H), 7.52ꢀ7.41 (m, 5H), 7.24 (d, J = 8.10 Hz, 1H), 7.17 (t, J = 8.02 Hz,

1H), 7.07 (br s, 1H), 3.99ꢀ3.76 (m, 4H), 3.74ꢀ3,37 (m, 4H), 2.00 (br s,

3H). HRMS calcdforC₂₂H₂₃N₇O₂(Mþ H)^þ418.1986, found 418.1993.

N-(3-(4-(4-Phenoxypiperidin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (35). The title compound was prepared using a

method analogous to the preparation of 27. Crude solutions were

purified with preparative RP-HPLC using CH₃CN (with 0.1% trifluor-

oacetic acid) in trifluoroacetic acid (0.1% aqueous) as the eluent. Frac-

tions containing the product were combined and washed with saturated

NaHCO₃. The product was extracted with CH₂Cl₂. The organic phase

was washed with brine, dried over MgSO₄, filtered, and concentrated,

providing the title compound as an off-white solid. Yield: 59 mg, 38%.

LCꢀMS (ESI) m/z: 405.0 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ

9.85 (s, 1H), 9.59 (s, 1H), 8.23 (s, 1H), 8.10 (br s, 1H), 7.33ꢀ7.24 (m,

3H), 7.20ꢀ7.08 (m, 2H), 7.03ꢀ6.97 (m, 2H), 6.93 (tt, J = 1.05, 7.31 Hz,

1H), 4.67 (tt, J = 3.83, 7.57 Hz, 1H), 4.27ꢀ4.11 (m, 2H), 3.61 (t, J = 9.63

Hz, 2H), 2.05ꢀ1.93 (m, 5H), 1.70ꢀ1.54 (m, 2H). HRMS calcd for

C₂₂H₂₄N₆O₂(M þ H)^þ405.2034, found 405.2036.

N-(3-(4-(4-(Benzyloxy)piperidin-1-yl)-1,3,5-triazin-2-ylamino)-

phenyl)acetamide (36). The title compound was prepared using a

method analogous to the preparation of compound 27. The crude

The crude material was dissolved in CH₂Cl₂(10 mL), and TFA was

added (1 mL). The mixture was stirred at room temperature for 18 h.

The mixture was dried under reduced pressure and puriﬁed using catch

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and release with a 5 g SCX-2 column, washing ﬁrst with CH₃OH, then

with 2 M NH₃in CH₃OH to elute basic materials. The light brown oil

isolated contained small quantities of product, 4-(1-phenylethoxy)-

piperidine, along with multiple impurities. It was used in the subsequent

step without further puriﬁcation. LCꢀMS (ESI) m/z: 206.2 (M þ 1).

To a solution of 4-(1-phenylethoxy)piperidine (240 mg, 1169 μmol)

in N,N-dimethylformamide (3.9 mL, 1169 μmol) was added N-(3-(4-

chloro-1,3,5-triazin-2-ylamino)-2-methylphenyl)acetamide (325 mg,

1169 μmol) followed by N,N-diisopropylethylamine (204 μL, 1169

μmol). The resulting solution was shaken at room temperature for 3 h.

The mixture was dried under reduced pressure and puriﬁed by silica gel

chromatography (0ꢀ100% CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in

CH₂Cl₂), providing product along with 25% impurity. The material

was repuriﬁed with preparative RP-HPLC using CH₃CN (with 0.1%

triﬂuoroacetic acid) in triﬂuoroacetic acid (0.1% aqueous) as the eluent.

Fractions containing the product were combined and washed with

saturated NaHCO₃. The product was extracted with ethyl acetate (2ꢁ).

The organic phasewas washedwith brine, dried overNa₂SO₄, ﬁltered, and

concentrated, providing the title compound. The oil obtained was

lyophilized from CH₃OH/H₂O to provide the title compound (1.2 mg,

0.2%) as a white powder. LCꢀMS (ESI) m/z: 447.2 (M þ 23). ¹H NMR

(400 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.96 (s, 1H), 8.09 (s, 1H),

7.38ꢀ7.31 (m, 4H), 7.29ꢀ7.22 (m, 1H), 7.20ꢀ7.06 (m, 3H), 4.66 (q, J =

6.42 Hz, 1H), 4.22ꢀ3.86 (m, 2H), 3.52ꢀ3.38 (m, 1H), 3.26 (d, J = 5.97

Hz, 2H), 2.47ꢀ2.43 (m, 1H), 2.04 (s, 3H), 2.02 (s, 3H), 1.92ꢀ1.81 (m,

1H), 1.72ꢀ1.60 (m, 1H), 1.52ꢀ1.37 (m, 1H), 1.32 (s, 3H).

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 20 mg, 10%. LCꢀMS (ESI)

m/z: 501.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H),

8.97 (s, 1H), 8.12 (s, 1H), 7.77ꢀ7.64 (m, 3H), 7.55ꢀ7.47 (m, 1H),

7.21ꢀ7.04 (m, 3H), 4.70 (s, 2H), 4.21ꢀ3.92 (m, 2H), 3.71 (tt, J = 3.81,

7.73 Hz, 1H), 3.47ꢀ3.32 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.94ꢀ1.83

(m, 2H), 1.54ꢀ1.42 (m, 2H). HRMS calcd for C₂₅H₂₇F₃N₆O₂(M þ

H)^þ501.222, found 501.2226.

N-(2-Methyl-3-(4-(4-(3-(trifluoromethyl)benzyloxy)piperidin-

1-yl)-1,3,5-triazin-2-ylamino)phenyl)acetamide (45). The title

compound was prepared using a method analogous to the preparation of

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 47 mg, 23%. Purity: 94%.

LCꢀMS (ESI) m/z: 501.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ

9.35 (s, 1H), 8.98 (s, 1H), 8.12 (s, 1H), 7.72ꢀ7.50 (m, 4H), 7.22ꢀ7.03

(m, 3H), 4.64 (s, 2H), 4.22ꢀ3.91 (m, 2H), 3.72ꢀ3.64 (m, 1H),

3.48ꢀ3.34 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.95ꢀ1.82 (m, 2H),

1.56ꢀ1.37 (m, 2H). HRMS calcd for C₂₅H₂₇F₃N₆O₂(M þ H)^þ

501.222, found 501.2224.

N-(2-Methyl-3-(4-(4-(4-(trifluoromethyl)benzyloxy)piperidin-

1-yl)-1,3,5-triazin-2-ylamino)phenyl)acetamide (46). The title

compound was prepared using a method analogous to the preparation of

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 634 mg, 62%. LCꢀMS

(ESI) m/z: 501.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.37 (s,

1H), 9.01 (s, 1H), 8.12 (s, 1H), 7.71 (d, J = 8.02 Hz, 2H), 7.57 (d, J =

7.92 Hz, 2H), 7.20ꢀ7.06 (m, 3H), 4.65 (s, 2H), 4.22ꢀ3.92 (m, 2H),

3.72ꢀ3.59 (m, 1H), 3.52ꢀ3.34 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H),

1.96ꢀ1.79 (m, 2H), 1.56ꢀ1.39 (m, 2H). HRMS calcd for C₂₅H₂₇F_3-

N₆O₂(M þ H)^þ501.222, found 501.2221.

N-(3-(4-(4-(2-Fluorobenzyloxy)piperidin-1-yl)-1,3,5-triazin-

2-ylamino)-2-methylphenyl)acetamide (41). The title com-

pound was prepared using a method analogous to the preparation of

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 66 mg, 52%. LCꢀMS (ESI)

m/z: 451.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H),

8.96 (s, 1H), 8.11 (s, 1H), 7.47 (dt, J = 1.76, 7.63 Hz, 1H), 7.42ꢀ7.32 (m,

1H), 7.22ꢀ7.05 (m, 5H), 4.61ꢀ4.53 (m, 2H), 4.18ꢀ3.92 (m, 2H),

3.72ꢀ3.60 (m, 1H), 3.48ꢀ3.31 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H),

1.93ꢀ1.81 (m, 2H), 1.53ꢀ1.39 (m, 2H). HRMS calcd for C₂₄H₂₇FN₆O₂

(M þ H)^þ451.2252, found 451.2255.

N-(2-Methyl-3-(4-(4-(4-methylbenzyloxy)piperidin-1-yl)-

1,3,5-triazin-2-ylamino)phenyl)acetamide (47). The title com-

pound was prepared using a method analogous to the preparation of 52.

Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 45 mg, 19%. LCꢀMS (ESI)

m/z: 447.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (s, 1H),

8.97 (s, 1H), 8.11 (s, 1H), 7.26ꢀ7.20 (m, 2H), 7.19ꢀ7.06 (m, 5H), 4.48

(s, 2H), 4.17ꢀ3.92 (m, 2H), 3.66ꢀ3.56 (m, 1H), 3.34 (br s, 2H),

2.32ꢀ2.25 (m, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.93ꢀ1.78 (m, 2H),

1.54ꢀ1.36 (m, 2H). HRMS calcd for C₂₅H₃₀N₆O₂(M þ H)^þ

447.2503, found 447.2504.

N-(3-(4-(4-(4-Isopropylbenzyloxy)piperidin-1-yl)-1,3,5-

triazin-2-ylamino)-2-methylphenyl)acetamide (48). The title

compound was prepared using a method analogous to the preparation of

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 69 mg, 27%. LCꢀMS (ESI)

m/z: 475.3 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (s, 1H),

8.97 (s, 1H), 8.11 (s, 1H), 7.29ꢀ7.07 (m, 7H), 4.52 (s, 2H), 4.17ꢀ3.88

(m, 2H), 3.67ꢀ3.58 (m, 1H), 3.47ꢀ3.34 (m, 2H), 2.87 (spt, J = 6.90 Hz,

1H), 2.04 (s, 3H), 2.03 (s, 3H), 1.91ꢀ1.80 (m, 2H), 1.50ꢀ1.40 (m, 2H),

1.19 (d, J = 6.94 Hz, 6H). HRMS calcd for C₂₇H₃₄N₆O₂(M þ H)^þ

475.2816, found 475.2817.

N-(3-(4-(4-(3-Fluorobenzyloxy)piperidin-1-yl)-1,3,5-triazin-

2-ylamino)-2-methylphenyl)acetamide (42). The title com-

pound was prepared using a method analogous to the preparation of

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 90 mg, 69%. LCꢀMS (ESI)

m/z: 451.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H),

8.96 (s, 1H), 8.11 (s, 1H), 7.42ꢀ7.35 (m, 1H), 7.22ꢀ7.13 (m, 4H),

7.13ꢀ7.04 (m, 2H), 4.56 (s, 2H), 3.93ꢀ4.19 (m, 2H), 3.70ꢀ3.60 (m,

1H), 3.52ꢀ3.32 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.92ꢀ1.82 (m, 2H),

1.55ꢀ1.38 (m, 2H). HRMS calcd for C₂₄H₂₇FN₆O₂(M þ H)^þ

451.2252, found 451.2253.

N-(3-(4-(4-(4-Fluorobenzyloxy)piperidin-1-yl)-1,3,5-triazin-

2-ylamino)-2-methylphenyl)acetamide (43). The title compound

was prepared using a method analogous to the preparation of 52. Crude

solutions were purified with preparative mass triggered HPLC using

CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the eluent,

providing the title compound. Yield: 75 mg, 58%. LCꢀMS (ESI) m/z:

451.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.96 (s,

1H), 8.11 (s, 1H), 7.43ꢀ7.32 (m, 2H), 7.22ꢀ7.04 (m, 5H), 4.52 (s, 2H),

4.19ꢀ3.89 (m, 2H), 3.69ꢀ3.58 (m, 1H), 3.48ꢀ3.32 (m, 2H), 2.04 (s,

3H), 2.03 (s, 3H), 1.91ꢀ1.78 (m, 2H), 1.53ꢀ1.34 (m, 2H). HRMS calcd

for C₂₄H₂₇FN₆O₂(M þ H)^þ451.2252, found 451.2254.

N-(3-(4-(4-(4-tert-Butylbenzyloxy)piperidin-1-yl)-1,3,5-

triazin-2-ylamino)-2-methylphenyl)acetamide (49). The title

compound was prepared using a method analogous to the preparation of

52. Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 109 mg, 56%. LCꢀMS

(ESI) m/z: 489.4 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (s,

N-(2-Methyl-3-(4-(4-(2-(trifluoromethyl)benzyloxy)piperidin-

1-yl)-1,3,5-triazin-2-ylamino)phenyl)acetamide (44). The title

compound was prepared using a method analogous to the preparation of

4441

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Journal of Medicinal Chemistry

ARTICLE

1H), 8.97 (s, 1H), 8.11 (s, 1H), 7.38ꢀ7.33 (m, 2H), 7.31ꢀ7.22 (m, J =

8.30 Hz, 2H), 7.21ꢀ7.05 (m, 3H), 4.52 (s, 2H), 4.17ꢀ3.93 (m, 2H),

3.63 (tt, J = 3.79, 7.75 Hz, 1H), 3.47ꢀ3.33 (m, 2H), 2.04 (s, 3H), 2.03 (s,

3H), 1.90ꢀ1.81 (m, 2H), 1.52ꢀ1.40 (m, 2H), 1.29 (s, 9H). HRMS

calcd for C₂₈H₃₆N₆O₂(M þ H)^þ489.2973, found 489.2973.

LCꢀMS (ESI) m/z: 463.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆)

δ 9.34 (s, 1H), 8.96 (s, 1H), 8.11 (s, 1H), 7.29ꢀ7.23 (m, 2H), 7.20ꢀ7.14

(m, 2H), 7.13ꢀ7.07 (m, 1H), 6.93ꢀ6.86 (m, 2H), 4.45 (s, 2H),

4.18ꢀ3.92 (m, 2H), 3.74 (s, 3H), 3.66ꢀ3.56 (m, 1H), 3.44ꢀ3.34 (m,

2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.89ꢀ1.79 (m, 2H), 1.52ꢀ1.34 (m, 2H).

HRMS calcd for C₂₅H₃₀N₆O₃(M þ H)^þ463.2452, found 463.2457.

N-(2-Methyl-3-(4-(4-(4-(trifluoromethoxy)benzyloxy)-

piperidin-1-yl)-1,3,5-triazin-2-ylamino)phenyl)acetamide

(52). To a dried flask charged with tert-butyl 4-hydroxypiperidine-1-

carboxylate (3.60 g, 17.9 mmol) was added N,N-dimethylformamide

(40 mL), and the resulting solution was cooled in an iceꢀwater bath

prior to the addition of sodium hydride (60%) (748 mg, 18.9 mmol) in

two portions 5 min apart. The mixture was stirred for 1 h and allowed to

warm to room temperature over that time. The resulting suspension was

cooled in an iceꢀwater bath prior to the addition of a solution of

1-(bromomethyl)-4-(trifluoromethoxy)benzene (5.018 g, 19.7 mmol)

in DMF (5 mL) via syringe. The resulting mixture was allowed to slowly

warm to room temperature while stirring for 15 h. The resulting pale

yellow suspension was carefully diluted with water (∼150 mL) and

extracted with ethyl acetate (2ꢁ). The combined organics were dried

with Na₂SO₄, filtered, and dried under reduced pressure. The material

was purified by silica gel chromatography (0ꢀ50% ethyl acetate in

hexanes), providing tert-butyl 4-(4-(trifluoromethoxy)benzyloxy)-

piperidine-1-carboxylate a white crystalline solid upon drying (5 g,

13.3 mmol). LCꢀMS (ESI) m/z: 398.2 (M þ 23).

4-((1-(4-(3-Acetamido-2-methylphenylamino)-1,3,5-triazin-

2-yl)piperidin-4-yloxy)methyl)benzamide (50). The title com-

pound was prepared using a method analogous to the preparation of 52.

Crude solutions were purified with preparative mass triggered HPLC

using CH₃CN (with 0.1% NH₄OH) in NH₄OH (0.1% aqueous) as the

eluent, providing the title compound. Yield: 1.2 mg, 0.5%. LCꢀMS

1

(ESI) m/z: 475.3 (M^þ). H NMR (400 MHz, DMSO-d₆) δ 9.33 (s,

1H), 8.95 (s, 1H), 8.11 (s, 1H), 7.90 (br s, 1H), 7.85 (d, J = 8.22 Hz, 2H),

7.40 (d, J = 8.31 Hz, 2H), 7.32ꢀ7.25 (m, 1H), 7.20ꢀ7.14 (m, 2H),

7.14ꢀ7.08 (m, 1H), 4.59 (s, 2H), 4.19ꢀ3.94 (m, 2H), 3.69ꢀ3.61 (m,

1H), 3.44ꢀ3.34 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.93ꢀ1.82 (m, 2H),

1.54ꢀ1.41 (m, 2H). HRMS calcd for C₂₅H₂₉N₇O₃(M þ H)^þ476.2405,

found 476.2406.

N-(3-(4-(4-(4-Methoxybenzyloxy)piperidin-1-yl)-1,3,5-

triazin-2-ylamino)-2-methylphenyl)acetamide (51). To a solu-

tion of tert-butyl 4-hydroxypiperidine-1-carboxylate (1.00 g, 4.97 mmol)

in N,N-dimethylformamide (19.9 mL) cooled in an iceꢀwater bath was

added NaH (0.219 g, 5.47 mmol). The bath was removed and the mix-

ture stirred for 2 h at room temp, providing a cloudy yellow suspension.

The resulting yellow suspension was cooled in an iceꢀwater bath, and

1-(chloromethyl)-4-methoxybenzene (1.01 mL, 7.45 mmol) was added

over 15 min. The mixture was allowed to stir for 4 h while slowly

warming to room temperature. To the turbid yellow solution was

carefully added water, and the resulting mixture was extracted with

ethyl acetate (2ꢁ). The combined organic washes were dried with

Na₂SO₄, filtered, and dried under reduced pressure. The crude yellow oil

obtained was purifiedbysilica gel chromatography(0ꢀ100%ethylacetate

in hexanes), providing tert-butyl 4-(4-methoxybenzyloxy)piperidine-1-

carboxylate (935 mg, 2.91 mmol, 58.5% yield) as a colorless oil. LCꢀMS

(ESI) m/z: 344.4 (M þ 23).

tert-Butyl 4-(4-(triﬂuoromethoxy)benzyloxy)piperidine-1-carboxy-

late (5 g, 13.3 mmol) was dissolved in CH₂Cl₂(40 mL), and triﬂuor-

oacetic acid (4 mL) was added. The light yellow solution was stirred at

room temperature for 20 h. The solution was dried under reduced

pressure, providing 4-(4-(triﬂuoromethoxy)benzyloxy)piperidine tri-

ﬂuoroacetate as a pale yellow oil (5.89 g, 85% (two steps)). LCꢀMS

(ESI) m/z: 276.2 (M þ 1).

A solution of 4-(4-(triﬂuoromethoxy)benzyloxy)piperidine triﬂuor-

oacetate (1.90 g, 4.9 mmol) in N,N-dimethylformamide (12.0 mL, 3.60

mmol) was cooled in an iceꢀwater bath prior to the addition of N,N-

diisopropylethylamine (2.08 mL, 12.0 mmol) and N-(3-(4-chloro-1,3,5-

triazin-2-ylamino)-2-methylphenyl)acetamide (1.00 g, 3.60 mmol),

respectively. The mixture was allowed to warm slowly to room tem-

perature and was stirred for 4 h. The material was dried under reduced

pressure and the resulting residue triturated with CH₂Cl₂/MeOH

(∼1:1), providing a pale yellow solid which contained a minor impurity.

The material was dissolved in 20 mL of 1:1 MeOH/DCM and sonicated

for approximately 1 min. Puriﬁcation was performed on a Prep SFC-2

using the following conditions: 25:75:0.2 CH₃OH/CO₂/diethylamine

on a 20 mm ꢁ 250 mm, 5 μm ChiralPak AD-H column at 80 mL/min

and 100 bar system pressure. Thirty injections were automated based on

the 0.75 mL pilot injection, providing the title compound (1.45 g, 78.0%

yield) as a white solid. LCꢀMS (ESI) m/z: 517.2 (M þ 1). ¹H NMR

(400 MHz, DMSO-d₆) δ 9.37 (s, 1H), 9.01 (s, 1H), 8.12 (s, 1H), 7.47

(d, J = 8.80 Hz, 2H), 7.33 (d, J = 7.73 Hz, 2H), 7.21ꢀ7.14 (m, 2H),

7.12ꢀ7.06 (m, 1H), 4.57 (s, 2H), 4.19ꢀ3.91 (m, 2H), 3.72ꢀ3.60 (m,

1H), 3.48ꢀ3.36 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H), 1.95ꢀ1.80 (m, 2H),

1.55ꢀ1.36 (m, 2H). HRMS calcd for C₂₅H₂₇F₃N₆O₃(M þ H)^þ

517.2169, found 517.2182. See Supporting Information for peak assign-

ments and proton and carbon spectra.

To a solution of tert-butyl 4-(4-methoxybenzyloxy)piperidine-1-

carboxylate (300 mg, 0.933 mmol) in CH₃OH (1.9 mL) was added 2

N NaOH (1.9 mL) in a 20 mL microwave vial. The vessel was sealed and

irradiated for 40 min at 165 ꢀC. The resulting mixture was diluted with

water and extracted with ethyl acetate (2ꢁ). The combined organics

were dried with Na₂SO₄, ﬁltered, and dried under reduced pressure. The

crude oil was ﬁltered through a 5 g SCX-2 column, washing ﬁrst with

MeOH, then with 2 M NH₃in MeOH, providing 4-(4-methoxybenzy-

loxy)piperidine (180 mg, 0.813 mmol, 87% yield) as a yellow oil. LCꢀ

MS (ESI) m/z: 222.2 (M þ 1).

To a ﬂask charged with N-(3-(4-chloro-1,3,5-triazin-2-ylamino)-2-

methylphenyl)acetamide (226 mg, 0.813 mmol) was added N,N-

dimethylformamide (3.25 mL) followed by N,N-diisopropylethylamine

(156 μL, 0.895 mmol) and 4-(4-methoxybenzyloxy)piperidine (180 mg,

0.813 mmol). The mixture was stirred for 16 h at room temperature. The

mixture was dried under reduced pressure and the residue triturated with

CH₂Cl₂, providing a yellow solid which was collected by vacuum ﬁltration

and washed with excess CH₂Cl₂. The ﬁltrate was dried under reduced

pressure and the oil obtained and puriﬁed by silica gel chromatography

(0ꢀ100% CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in CH₂Cl₂), leading to

the isolation mixture of two compounds primarily by TLC, conﬁrmed by

LCꢀMS. The solid obtained (∼65 mg) was repuriﬁed with preparative

RP-HPLC using CH₃CN (with 0.1% triﬂuoroacetic acid) in triﬂuoroace-

tic acid (0.1% aqueous) as the eluent. A small portion of the material was

obtained because of instrument error. The material obtained was free-

based using a 2 g SCX-2 column, washing ﬁrst with CH₃OH, then with 2

M NH₃in CH₃OH. The basic wash was collected, dried under reduced

pressure, and the oil obtained was lyophilized from CH₃OH/water to

provide the title compound (5 mg, 1.3% yield) as a white powder.

N-(2-Methyl-3-(4-(4-(4-(trifluoromethyl)phenoxy)piperidin-

1-yl)-1,3,5-triazin-2-ylamino)phenyl)acetamide (53). The title

compound was prepared using a method analogous to the preparation of

21 using precursor 22. Crude solutions were purified with preparative

mass triggered HPLC using CH₃CN (with 0.1% NH₄OH) in NH₄OH

(0.1% aqueous) as the eluent, providing the title compound. Yield: 77

1

mg, 32%. LCꢀMS (ESI) m/z: 487.2 (M þ 1). H NMR (400 MHz,

DMSO-d₆) δ 9.35 (s, 1H), 9.03 (s, 1H), 8.13 (s, 1H), 7.64 (d, J = 8.61

Hz, 2H), 7.23ꢀ7.14 (m, 4H), 7.13ꢀ7.07 (m, 1H), 4.82ꢀ4.74 (m, 1H),

4442

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Journal of Medicinal Chemistry

ARTICLE

4.23ꢀ3.95 (m, 2H), 3.63ꢀ3.41 (m, 2H), 2.04 (s, 3H), 2.03 (s, 3H),

2.01ꢀ1.93 (m, 2H), 1.65ꢀ1.54 (m, 2H). HRMS calcd for C₂₄H₂₅F₃N₆O₂

(M þ H)^þ487.2064, found 487.2064.

N-diisopropylethylamine (17 μL, 0.13 mmol) in N,N-dimethylforma-

mide (0.5 mL). The resulting mixture was stirred at room temperature

for 30 min, then dried under reduced pressure. The crude material was

purified with reverse phase HPLC (C18, mobile phase = 40% CH₃CN,

UV detection = 241 nm, flow = 6 mL/min), providing product with

specific activity of 54.4 Ci/mmol, 99% purity.

N-(2-Methyl-3-(4-(4-(4-(trifluoromethoxy)phenoxy)piperidin-

1-yl)-1,3,5-triazin-2-ylamino)phenyl)acetamide (54). The title

compound was prepared using a method analogous to the preparation of

21 using precursor 22. Crude solutions were purified with preparative

mass triggered HPLC using CH₃CN (with 0.1% NH₄OH) in NH₄OH

(0.1% aqueous) as the eluent, providing the title compound. Yield: 58

’ ASSOCIATED CONTENT

1

mg, 64%. LCꢀMS (ESI) m/z: 503.2 (M þ 1). H NMR (400 MHz,

S

Supporting Information.

Additional diagrams and

b

DMSO-d₆) δ 9.36 (s, 1H), 9.03 (s, 1H), 8.14 (s, 1H), 7.28 (dd, J = 0.68,

9.10 Hz, 2H), 7.21ꢀ7.14 (m, 2H), 7.14ꢀ7.05 (m, 3H), 4.65 (tt, J = 3.61,

7.64 Hz, 1H), 4.20ꢀ4.00 (m, 2H), 3.61ꢀ3.41 (m, 2H), 2.04 (s, 6H),

2.00ꢀ1.90 (m, 2H), 1.63ꢀ1.52 (m, 2H). HRMS calcd for C₂₄H₂₅F₃N₆O₃

(M þ H)^þ503.2013, found 503.2015.

tables illustrating details of state dependence, formalin, open

ﬁeld activity, radiolabeling studies, and NMR peak assignments

for 52. This material is available free of charge via the Internet at

http://pubs.acs.org.

N-(2-Methyl-3-(6-(4-(4-(trifluoromethoxy)benzyloxy)-

piperidin-1-yl)pyrimidin-4-ylamino)phenyl)acetamide (55).

To 4,6-dichloropyrimidine (1523 mg, 1.02 mmol) in 2-propanol (3 mL)

at ambient temperature was added N,N-diisopropylethylamine

(0.266 mL, 1.53 mmol) followed by 4-(4-(trifluoromethoxy)benzyloxy)-

piperidine (280 mg, 1.02 mmol). The resulting mixture was stirred at

room temperature for 3.5 h. The reaction mixture was concentrated

under reduced pressure and purified by silica gel chromatography (0ꢀ40%

ethyl acetate in hexanes), providing 4-chloro-6-(4-(4-(trifluoromethoxy)-

benzyloxy)piperidin-1-yl)pyrimidine (275 mg, 70%). LCꢀMS (ESI) m/z:

388.2 (M þ 1).

’ AUTHOR INFORMATION

Corresponding Author

*Phone: (617) 444-5351. Fax: (617) 621-3908. E-mail: hbregman@

amgen.com.

’ ACKNOWLEDGMENT

The authors thank Grace Bi, Matthew Potter, and Larry Miller

for puriﬁcation and analytical support, Paul H. Krolikowski for

NMR support, and Drs. Margaret Y. Chu-Moyer and Richard T.

Lewis for proofreading the manuscript.

Toa vialchargedwith 4-chloro-6-(4-(4-(triﬂuoromethoxy)benzyloxy)-

piperidin-1-yl)pyrimidine (105.6 mg, 0.272 mmol) and N-(3-amino-2-

methylphenyl)acetamide (52.2 mg, 0.300 mmol) were added 2-propa-

nol (2.5 mL) and triﬂuoroacetic acid (0.063 mL, 0.817 mmol). The

resulting mixture was heated at 73 ꢀC for 3 days, then at 90 ꢀC for 16

days. The reaction mixture was cooled to room temperature, dried under

reduced pressure, and puriﬁed with silica gel chromatography (0ꢀ80%

CH₂Cl₂/CH₃OH/NH₄OH (90:10:1) in CH₂Cl₂), providing the title

compound as a white solid. Yield: 22.7 mg, 16%. LCꢀMS (ESI) m/z:

516.2 (M þ 1). ¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.48 (br

s, 1H), 8.07 (s, 1H), 7.47 (d, J = 8.80 Hz, 2H), 7.33 (d, J = 7.92 Hz, 2H),

7.22ꢀ7.09 (m, 3H), 5.67 (s, 1H), 4.57 (s, 2H), 3.89ꢀ3.76 (m, 2H), 3.65

(tt, J = 4.08, 8.00 Hz, 1H), 3.24ꢀ3.13 (m, 2H), 2.05 (s, 3H), 2.03 (s,

3H), 1.93ꢀ1.83 (m, 2H), 1.54ꢀ1.42 (m, 2H). HRMS calcd for

C₂₆H₂₈F₃N₅O₃(M þ H)^þ516.2217, found 516.222.

’ ABBREVIATIONS USED

Nav1.7, voltage-gated sodium channel type 7; TTX, tetrodotox-

in; SCN9A, sodium channel, voltage-gated, type IX, R subunit;

SAR, structureꢀactivity relationship; DIEA, N,N-diisopropy-

lethylamine; TFA, triﬂuoroacetic acid; DMF, N,N-dimethylfor-

mamide;Boc, tert-butoxycarbonyl;SCX-2, strong cation exchange

2 (functionalized with propylsulfonic acid); hERG, human ether-

a-go-go-related gene; PK, pharmacokinetic; Cl_int, in vitro intrinsic

clearance; RLM, rat liver microsome; HLM, human liver micro-

some; F, oral bioavailability, PKDM, pharmacokinetics and drug

metabolism; K_R, aﬃnity for resting channels; KI, aﬃnity for

inactivated channel; DRG, dorsal root ganglion; K_V1.7, voltage-

gated potassium channel type 7; TRPM8, transient receptor

potential cation channel subfamily M, member 8; TRPV3,

transient receptor potential cation channel, subfamily V,

member 3; TRPV4, transient receptor potential cation channel,

subfamily V, member 4; P₂X₇, purinergic receptor P2X, ligand-

gated ion channel 7; GPCR, G-protein-coupled receptor; PX,

PatchXpress; BTX, batrachotoxin

³H-36. To a flask charged with 2,4-dichloro-1,3,5-triazine (5.0 g, 32

mmol) was added N,N-dimethylformamide (40 mL), and the mixture

was cooled to 0 ꢀC. To this solution were added N,N-diisopropylethy-

lamine (12 mL, 70 mmol) and 4-(benzyloxy)piperidine hydrochloride

(7.2 g, 32 mmol). The resulting mix was allowed to slowly warm for over

4 h. To the reaction mixture was added excess water, providing a yellow

precipitate which was collected via vacuum filtration and washed with excess

water followed by methanol, providing 2-(4-(benzyloxy)piperidin-1-yl)-4-

chloro-1,3,5-triazine (7.25 g, 75%). LCꢀMS (ESI) m/z: 305.1 (M þ 1).

To a mixture of 2-(4-(benzyloxy)piperidin-1-yl)-4-chloro-1,3,5-tria-

zine (4.72 g, 15.5 mmol) and benzene-1,3-diamine (2.09 g, 19.3 mmol)

in 2-propanol (50 mL) was added N,N-diisopropylethylamine (5.39 mL,

31 mmol). The resulting mixture was heated to 35 ꢀC for 29 h leading to

the formation of a precipitate which was collected via vacuum ﬁltration

and washed with excess 2-propanol, providing N¹-(4-(4-(benzyloxy)-

piperidin-1-yl)-1,3,5-triazin-2-yl)benzene-1,3-diamine (4.12 g, 71%

yield) as light brown solid upon drying under high vacuum.

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Radiolabel Incorporation Step. A solution of T-acetic acid

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Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain

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