Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors with activity in neurodegeneration models

Article

pubs.acs.org/jmc

Discovery of Dual Leucine Zipper Kinase (DLK, MAP3K12) Inhibitors

with Activity in Neurodegeneration Models

Snahel Patel,^†Frederick Cohen,^†,#Brian J. Dean,^§Kelly De La Torre,^†Gauri Deshmukh,^§

Anthony A. Estrada,^†Arundhati Sengupta Ghosh,^‡Paul Gibbons,^†Amy Gustafson,^∥Malcolm P. Huestis,^†

Claire E. Le Pichon,^‡,∞Han Lin,^‡Wendy Liu,^†Xingrong Liu,^§Yichin Liu,^∥Cuong Q. Ly,^†

Joseph P. Lyssikatos,^†,×Changyou Ma,^⊥Kimberly Scearce-Levie,^‡Young G. Shin,^§,○Hilda Solanoy,^‡

Kimberly L. Stark,^‡Jian Wang,^⊥Bei Wang,^‡,△Xianrui Zhao,^†Joseph W. Lewcock,*^,‡and Michael Siu*^,†

†

‡

§

∥

Departments of Discovery Chemistry, Neurosciences, Drug Metabolism and Pharmacokinetics, and Biochemical and Cellular

Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States

^⊥Department of Chemistry, WuXi AppTec Co., Ltd., 288 Fute Zhonglu, Wai Gao Qiao Free Trade Zone, Shanghai, 200131, P. R.

China

ABSTRACT: Dual leucine zipper kinase (DLK, MAP3K12)

was recently identiﬁed as an essential regulator of neuronal

degeneration in multiple contexts. Here we describe the

generation of potent and selective DLK inhibitors starting

from a high-throughput screening hit. Using proposed hinge-

binding interactions to infer a binding mode and speciﬁc

design parameters to optimize for CNS druglike molecules, we

came to focus on the di(pyridin-2-yl)amines because of their

combination of desirable potency and good brain penetration

following oral dosing. Our lead inhibitor GNE-3511 (26)

displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity

in two diﬀerent animal models of disease. These results suggest that speciﬁc pharmacological inhibition of DLK may have

therapeutic potential in multiple indications.

required for stress-induced JNK signaling in neurons.^13,14

Recent work from our group and others have identiﬁed the

importance of DLK as a central regulator of neuronal

degeneration in multiple contexts including models of

Parkinson’s disease, glaucoma/optic neuropathy, and excito-

toxic neurodegeneration, suggesting that this kinase may

INTRODUCTION

■

Neurodegenerative diseases, such as Alzheimer’s and Parkin-

son’s, represent signiﬁcant unmet medical needs, yet no

therapies exist that are able to slow the course of neuronal

loss. Consequently, considerable interest exists for molecules

that target the mechanisms underlying degeneration in these

represent an appealing target for treatment of these and related

and related contexts, as they represent an attractive approach

indications.¹⁵⁻²¹To date, however, only one study has

for treatment.^1,2For example, the c-Jun N-terminal kinase

examined the consequence of DLK inhibition using previously

(JNK) signaling pathway has received signiﬁcant attention in

this regard because of its proposed role in both acute and

chronic neurodegenerative paradigms. Genetic deletion or

identiﬁed compounds which also target other kinases.¹⁸In this

contribution, we describe the development of the ﬁrst potent

and selective DLK inhibitors derived from a high-throughput

inhibition of JNKs in neurons has been shown to be potently

neuroprotective in a number of settings,³⁻⁷and evidence of

screening eﬀort.

pathway activity exists in multiple indications ranging from

traumatic brain injury (TBI) to Alzheimer’s disease.^8,9On the

basis of these data, signiﬁcant eﬀort has been reported toward

the development of brain-penetrant JNK inhibitors for the

treatment of these indications,¹⁰but these endeavors have not

yet resulted in progression of compounds into the clinic,

highlighting the challenges associated with this approach.^11,12

We hypothesized that upstream regulators of JNK may exist

and targeting these regulators may provide an alternative

strategy to inhibiting JNK activity directly. The MAP3K DLK

(dual leucine zipper kinase) appears to be an ideal candidate for

this role, as it displays neuronal-speciﬁc expression and is

RESULTS AND DISCUSSION

■

In order to identify novel small molecule DLK inhibitors, our

internal compound collection was screened against the kinase

domain of DLK.²²For the primary screen, percent inhibition

was measured at a compound concentration of 5 μM. For

compounds that showed >50% inhibition, half-maximal

Special Issue: New Frontiers in Kinases

Received: September 10, 2014

A

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inhibitory concentrations were determined. This assay was also

used for characterization of subsequent hit optimization eﬀorts

followed by a cell-based assay that measured levels of JNK

phosphorylation (p-JNK) induced by DLK overexpression. On

the basis of these activity measurements and physicochemical

properties, N-(pyrimidin-4-yl)thiazol-2-amine 1 (Figure 1) was

Schrodinger’s Jaguar at the B3LYP/6-31G** level of theory

conﬁrmed that the proposed planar conformation was the

lowest energy conformation by over 1.7 kcal/mol.^35,36As a

result of this low energy conformation, the piperidine

benzamide and methyl groups are oriented as depicted in

Figure 2 toward solvent and the P-loop regions, respectively.

On the basis of this proposed binding mode, we evaluated 2-

aminopyridines as 2-aminothiazole bioisosteric replacements.

Either a 2-amino-4-methylpyridine or 2-amino-5-methylpyr-

idine could potentially overlay with the 2-amino-5-methyl-

thiazole of 1. Upon experimental evaluation, the matched pairs

2-amino-5-methylpyridine 2 (K_i= 0.401 μM)/2-amino-4-

methylpyridine 3 (K_i= 0.085 μM) and 2-amino-5-triﬂuor-

omethylpyridine 4 (K_i= 0.691 μM)/2-amino-4-triﬂuorome-

thylpyridine 5 (K_i= 0.024 μM) proved the C4 position to be

the most optimal for potency (Table 1). Despite the decrease

in lipophilic ligand eﬃciency (LipE) on going from an

aminothiazole to an aminopyridine, the removal of a potential

toxicophore was realized.³⁷The consistent LipE (∼3) of the

C4-alkylpyridine analogs in Table 1 suggested that the

Figure 1. HTS hit 1.

selected for optimization. Inhibitor 1 had acceptable potency

(DLK K_i= 0.035 μM, p-JNK IC₅₀= 0.641 μM), permeability

(MDR1-MDCK BA/AB = 2.2, AB = 4.3 × 10⁻⁶cm/s), ligand

eﬃciency²³(LE = 0.37), and lipophilic ligand eﬃciency^24,25

(LipE = 3.6). The property space of inhibitor 1 (ClogP and

ClogD = 3.9, tPSA = 71 Å², cpK_a= 6.8, HBD = 1, MW = 398,

CNS MPO = 4.2) was acceptable for optimization of a kinase

inhibitor for brain penetration.^26,27In addition to improvement

in potency, selectivity against the JNK pathway kinases (DLK

→ MKK4/7 → JNK2/3 → cJun) and MLK 1/2/3 (kinases

most related to DLK)²⁸is also necessary before in vivo

evaluation. For initial SAR development, alternatives to the

literature prevalent kinase hinge-binding 2-amino-5-substituted

thiazole motif^29,30were examined because of concerns about

selectivity and metabolic liabilities of this group (reactive

metabolite formation).³¹

a

Table 1. Heteroarylamine SAR

With no crystal structures of DLK in the public domain to

guide initial SAR, we proposed a binding mode for compound

1, as outlined in Figure 2, where the 2-aminothiazole binds to

Figure 2. Proposed binding mode of HTS hit 1 in DLK.

the kinase domain hinge residues via two hydrogen-bonding

interactions and a nonclassical thiazole C4−H interaction.³²

The favorable electrostatic pyrimidine N3 to thiazole S1

interaction was proposed to orient these rings coplanar.^33,34

Generation of reasonable low energy conformations using

OpenEye’s OMEGA tool of the N-(2,6-dimethylpyrimidin-5-

yl)-5-methylthiazol-2-amine substructure of compound 1 and

quantum mechanical minimization of resultant structures using

a

Compounds were tested as racemates. All assay results represent the

geometric mean of a minimum of two determinations, and these assays

generally produced results within 3-fold of the reported mean. LipE =

−log K_i− ClogP.

b

B

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a

Table 2. 2- and 6-Pyrimidine Substitution

a

Compounds 12−14 were tested as racemates. All assay results represent the geometric mean of a minimum of two determinations, and these assays

b

c

d

generally produced results within 3-fold of the reported mean. Liver microsome-predicted hepatic clearance. H/R/M = human/rat/mouse. LipE

= −log K_i− ClogP.

improvement in observed potency was due to lipophilicity

instead of speciﬁc interactions between this position of the

ligand with DLK (i.e., methylpyridine 3 LipE = 2.9 and

triﬂuoromethylpyridine 5 LipE = 2.9). Further evaluation of C4

substituents showed isopropyl 7, cyclopropyl 8, cyano 10, and

methoxy 11 were tolerated with the cyano being the most

lipophilic eﬃcient group (albeit with high tPSA). An

approximate 10- to 20-fold shift between the biochemical and

cellular assay was observed with these inhibitors, which is in

line with the ATP K_mreported for DLK.³⁸

mouse). Further potency exploration and an attempt to

increase microsomal stability of pyrrolidine 13 (due to

known soft spots)⁴¹led us to synthesize 3,3-diﬂuoropyrrolidine

at the C2 position of the pyrimidine. Selection of the gem-

ﬂuorinated moiety was based on its demonstrated ability to

improve pharmacokinetic proﬁle relative to mono- and

tetraﬂuorinated counterparts.^42,433,3-Diﬂuoropyrrolidine 14

maintained biochemical and cellular potency (K_i= 0.007 μM

and p-JNK IC₅₀= 0.408 μM) with good to moderate liver

microsome stability. Transposition of the 3-piperidine to the 4-

piperidine 15 eliminated the stereogenic center while

maintaining potency (K_i= 0.002 μM, p-JNK IC₅₀= 0.410

μM). These basic amines with two hydrogen bond donors were

eﬄux substrates in an in vitro MDR1−MDCK assay (14, eﬄux

ratio of 68). To reduce the basicity of the analogs and the

number of hydrogen bond donors, we examined the 4-

tetrahydropyran 16 and N-acetyl-4-piperidine 17, both of

which maintained reasonable potency; however, this potency

came at the expense of in vitro metabolic stability. Reducing

lipophilicity at the C2 position (by either removing a carbon or

introduction of a polar atom) resulted in the loss of

biochemical potency (3,3-diﬂuoroazetidine 18 K_i= 0.166 μM;

morpholine 19, K_i= 0.431 μM) with no apparent improvement

in metabolic stability.

Benzamide replacements with tPSA < 80 Å²(for CNS

penetration) proved to be unfruitful (not reported). Therefore,

we adopted a strategy to further optimize triﬂuoromethylpyr-

idine 5 by redistribution of the polarity and atoms of the

benzamide to examine the C2 position of the pyrimidine core.

For comparison, removal of the benzamide of 5 (compound 12,

Table 2) lost binding aﬃnity for DLK (∼30×). We surmised

that further hit optimization could be developed by growing the

vector presumably under the P-loop region.^39,40A cyclic

secondary amine was initially chosen at the C2 position of the

pyrimidine to avoid additional hydrogen bond donors on the

inhibitors that may diminish brain penetration properties.

Pyrrolidine 13 showed improved biochemical and cellular

potency (DLK K_i= 0.007 μM, p-JNK IC₅₀= 0.109 μM) with

moderate stability in liver microsomes (human, rat, and

C

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In an eﬀort to modify the central core to reduce tPSA, a

pyridine scaﬀold was explored. Our initial eﬀorts focused on

changing the 2,4-diaminopyrimidine to a 2,6-diaminopyridine

scaﬀold in order to maintain an overall planar conformation

(Figure 3). The initial analog in this endeavor (20, Table 3)

examining the functional consequence of endogenous DLK

inhibition in neurons.¹⁴As expected, the IC₅₀for compound 20

in the axon degeneration assay was higher than the p-JNK IC₅₀

because near complete inhibition of DLK activity is likely

required to achieve neuroprotection in this context.

In order to further reduce lipophilicity of 20 and related

analogs, the previously identiﬁed polar and lipophilic eﬃcient 4-

cyano-2-aminopyridine hinge binder (Table 1) was used for

further exploration on the pyridine core. Cyanopyridine 21

(direct analog of 20) maintained lipophilic ligand eﬃciency

(LipE = 3.5) but introduced eﬄux. As in the case with the

pyrimidine scaﬀold, 4-tetrahydropyran 22 was tolerated and

gave rise to a potent inhibitor of DLK. The 4-cyano-4-

tetrahydropyran 23 improved metabolic stability, yet the

molecule possessed a high tPSA value. Truncated analogs of

23 that maintain a quaternary carbon center (24 and 25) were

potent and avoided eﬄux in an in vitro MDR1−MDCK assay

despite relatively high tPSA. The steric hindrance around the

tertiary nitrile 24 and tertiary alcohol 25 potentially reduces the

“eﬀective” polar surface area of these compounds.⁴⁴An

oxetane⁴⁵⁻⁴⁷was utilized to reduce basicity of the piperidine

in order to avoid eﬄux, and the resultant N-oxetanylpiperidine

GNE-3511 (26)²²proved to be one of the most cell potent

Figure 3. Core change to reduce tPSA while maintaining planar

conformation of inhibitors.

gave rise to an improvement in potency (K_i= 0.001 μM, p-JNK

IC₅₀= 0.069 μM) and lipophilic eﬃciency compared to

pyrimidine 17. Compound 20 also reached suﬃcient cellular

potency to elicit protection of primary rat dorsal root ganglion

(DRG) neurons in an in vitro axon degeneration assay with an

IC₅₀of 0.171 μM. This assay provides a method to further

characterize the activity of selected compounds through

a

Table 3. Pyridine Core C4-Substituent SAR

a

All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the

b

c

reported mean. MDCK−MDR1 human P-gp transfected cell line. Basolateral-to-apical/apical-to-basolateral. Units = ×10⁻⁶cm s⁻¹. Liver

microsome-predicted hepatic clearance. H/R/M = human/rat/mouse. LipE = −log K_i− ClogP.

d

e

D

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a

Table 4. Pyridine Core C6-Substituent SAR

a

All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the

b

c

reported mean. MDCK−MDR1 human P-gp transfected cell line. Basolateral-to-apical/apical-to-basolateral. Units = ×10⁻⁶cm s⁻¹. Liver

microsome-predicted hepatic clearance. H/R/M = human/rat/mouse. LipE = −log K_i− ClogP.

d

e

a

Table 5. Kinase Selectivity of Compound 26

IC₅₀(nM)

DLK K_i(nM)

MKK4

>5000

MKK7

>5000

JNK1

129

JNK2

514

JNK3

364

MLK1

67.8

MLK2

767

MLK3

602

<0.5

a

IC₅₀values were determined at Invitrogen.

inhibitors. The N-oxetanylazetidine 27 was equally potent

against DLK but was labile in microsomes despite a lower

ClogP. On the basis of these results, the 2-(6-(3,3-

diﬂuoropyrrolidin-1-yl)pyridin-2-ylamino)isonicotinonitrile

subscaﬀold clearly tolerated a variety of substituents on the

pyridine C4 position.

Examination of the C6 position of the pyridine was

continued with the 2-(4-(1-(oxetan-3-yl)piperidin-4-yl)-

pyridin-2-ylamino)isonicotinonitrile subscaﬀold (Table 4). 3-

Methoxyazetidine 28 and azetidine 29 lost potency with no

beneﬁt in metabolic stability or eﬄux properties (in the case of

28). In addition to amines, ethers such as the cyclobutyl ether

analog 30 maintained signiﬁcant potency (K_i= 0.0002 μM).

Larger alkyl groups provided increased binding aﬃnity for DLK

at this position (methyl 31 K_i= 0.093 μM, ethyl 32 K_i= 0.018

μM, and cyclopropyl 33 K_i= 0.001 μM).

Of the examples in Tables 3 and 4, compound 26 provided

the best balance of potency, in vitro metabolic stability, and

eﬄux properties. Inhibitor 26 had very good selectivity over the

other JNK pathway kinases and homologues of DLK (Table 5).

Compound 26 also exhibited very good overall kinase

selectivity in an Invitrogen panel of 298 kinases at 0.1 μM

(>200 × DLK K_i) (Figure 4). Lastly, inhibition of DLK by 26

translated to potent protection of primary neurons in an in

vitro axon degeneration assay with IC₅₀= 0.107 μM.

Pharmacokinetic evaluation revealed 26 exhibited moderate

(mouse, rat, and cynomolgus) to high (dog) in vivo plasma

clearances, moderate volumes of distribution, short half-lives,

and brain penetration suﬃcient to enable examination in animal

models of neurodegeneration (Table 6). DLK inhibitor 26 was

then tested in the mouse optic nerve crush model of axonal

injury, which mimics the degeneration that occurs in glaucoma

or optic neuropathy.^48,49Our previous studies demonstrated

that loss of DLK expression resulted in protection of retinal

ganglion cell neurons from degeneration as well as an

attenuation of downstream signaling following injury.^17,18In

this and other neuronal injury models, phosphorylation of c-Jun

(p-c-Jun) is strongly induced by injury in a DLK/JNK

dependent fashion and could thus be used as a pharmacody-

namic readout of DLK inhibition in vivo.^3,5,17Animals were

dosed orally with either inhibitor 26 at two dose levels or

vehicle control 30 min prior to nerve crush injury. Six hours

after insult, levels of p-c-Jun in retina were measured using a

MSD assay. Treatment with inhibitor 26 resulted in a dose-

dependent reduction of p-c-Jun present in retina (Figure 5).

To examine the activity of inhibitor 26 in brain and further

conﬁrm its ability to inhibit DLK activity in vivo, the MPTP (1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkin-

son’s disease was used.⁵⁰In this model, MPTP treatment

results in dramatic increases in p-c-Jun within the dopaminergic

neurons of the substantia nigra by 24 h which leads to the

E

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Figure 4. Kinase selectivity of compound 26. Invitrogen panel of 298 kinases. Kinases with >80% inhibition at 0.1 μM are EphA1, Lck, Src, STK16,

Syk, Flt3 (D835Y), Src_N1.

a

Table 6. PK Properties of DLK Inhibitor 26

b

c

species

mouse

CL_p(mL min⁻¹kg⁻¹

)

V_dss(L kg⁻¹

)

t_1/2(h)

F (%)

B_u/P_u

CSF/P_u

56

30

41

16

2.5

3.7

6.5

3.1

0.6

1.8

4

45

63

32

19

0.24 at 6 h

0.7

rat

0.4

dog

cynomolgous

2.4

0.6

a

b

Compounds were dosed iv (1 mg kg⁻¹) as a 60% PEG solution and po (5 mg kg⁻¹) as an aqueous suspension with 1% methylcellulose. unbound

c

brain/unbound plasma AUC ratio (unless noted otherwise). CSF/unbound plasma AUC ratio.

degeneration of these neurons at later time points.⁵To test the

CHEMISTRY

■

ability of 26 to reduce the induction of p-c-Jun, animals were

The preparation of compounds 1−12 was performed as shown

in Scheme 1 by reacting commercially available ( )-tert-butyl

3-(6-chloro-2-methylpyrimidin-4-yl)piperidine-1-carboxylate

(34, CAS no. 1361116-19-9) with the required heteroarylamine

via a Buchwald−Hartwig reaction.⁵¹⁻⁵³Subsequent acid-

mediated N-Boc deprotection resulted in a crude amine

which was directly coupled with benzoic acid. The syntheses

of 13−19 were carried out as outlined in Scheme 2. C6-

Substituted dichloropyrimidines tert-butyl 4-(2,6-dichloropyr-

imidin-4-yl)piperidine-1-carboxylate (39, CAS no. 1439823-01-

4) and 2,4-dichloro-6-(tetrahydro-2H-pyran-4-yl)pyrimidine

(40, CAS no. 1417519-39-1) are commercially available. The

synthesis of 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carbox-

ylic acid tert-butyl ester (38) commenced with the acylation of

( )-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (35)

with potassium monoethyl malonate to form ketoester 36

(CDI, MgCl₂, CH₃CN).⁵⁴Condensation of 36 with urea under

basic conditions produced 2,4-dihydroxypyrimidine 37, which

dosed with 37.5 or 75 mg/kg or vehicle 30 min prior to the

initiation of MPTP treatment and again at 12 and 24 h after the

ﬁrst dose in order to maintain necessary concentrations of

compound in brain. Brains were dissected at 1 h after the ﬁnal

dose of 26, and the number of p-c-Jun positive neurons was

measured. The high dose of inhibitor 26 resulted in a complete

suppression of c-Jun phosphorylation, while the low dose

reduced the number of p-c-Jun positive cells to an intermediate

level. Taken together, these data demonstrated that compound

26 is an orally bioavailable and brain-penetrant DLK inhibitor

with activity in multiple animal models of neurodegenerative

disease. This, along with the potent neuroprotective activity

observed with DLK inhibitor 26 in vitro, suggests that DLK

inhibition has attractive therapeutic potential.

F

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Figure 5. GNE-3511 (26) displays activity in two animal models of neurodegeneration. (A) Levels of p-c-Jun in retinal lysates were measured by

ELISA following nerve crush and treatment with inhibitor 26. Values are presented relative to uncrushed vehicle controls. N = 5 for vehicle and N =

6 for both inhibitor 26 treated groups: (∗∗∗) p < 0.001. (B) Number of p-c-Jun positive cells/mm²in animals following MPTP treatment with or

without inhibitor 26 treatment as compared to controls. N = 8/group from two cohorts: (∗∗) p < 0.01, (∗∗∗) p < 0.01. For both panels, bars

represent the mean and error bars represent SEM. Dots represent data points from individual animals in the study. In panel B, circles and triangles

represent animals from independent cohorts.

a

The pyridine analogs (Tables 3 and 4) were synthesized

according to the synthetic routes shown in Schemes 3−7. The

majority of the pyridine analogs were synthesized from 2,6-

dichloro-4-iodopyridine (43) using various metal-mediated

cross-coupling reactions. Suzuki−Miyaura cross coupling of

N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester

(44) with 2,6-dichloro-4-iodopyridine (43, Scheme 3) provided

an intermediate pyridine suitable for additional substitution

with 3,3-diﬂuoropyrrolidine via S_NAr displacement followed by

a Buchwald−Hartwig amination with 2-amino-4-triﬂuorome-

thypyridine. The resulting alkene 47 was reduced through

hydrogenation. Protecting group removal followed by acylation

with acetyl chloride aﬀorded 20. To circumvent an alkene

reduction, Negishi cross coupling of 2,6-dichloro-4-iodopyr-

idine (43) with N-Boc 4-iodopiperidine (50, Scheme 4)

provided the intermediate 2,6-dichloropyridine 51.⁵⁵From this

intermediate, S_NAr with 3,3-diﬂuoropyrrolidine provided the

corresponding 4-substituted 2-amino-6-chloropyridine 52. N-

tert-Butoxycarbonyl deprotection and subsequent reductive

amination with oxetan-3-one provided the N-oxetanylpiper-

idine 53.⁵⁶Compound 26 was obtained from chloropyridine 53

via Buchwald−Hartwig amination with 2-amino-4-cyanopyr-

idine. Alternatively, initial introduction of the oxetane (or

acetyl) followed by 2-amino-4-cyanopyridine provided inter-

mediate 54 which allowed for late-stage palladium catalyzed

Buchwald−Hartwig amination⁴⁹⁻⁵¹(compounds 21, 28, 29),

Beller etheriﬁcation⁵⁷(ether 30), and B-alkyl Suzuki

coupling^58,59(ethyl 32 and cyclopyropyl 33). Tetrahydropyran

22 and N-oxetanylazetidine 27 were obtained using the

reaction sequence in Scheme 5 from an initial Negishi coupling

of 4-iodotetrahydropyran and N-Boc-3-iodoazetidine, respec-

tively. Introduction of a quaternary nitrile at the C4 of pyridines

occurred via regioselective addition of α-lithiated nitriles to

2,4,6-trichloropyridine (61) to provide 4-substituted-2,6-

dichloropyridines 62 and 63 (Scheme 6).⁶⁰⁻⁶³From these

intermediates, sequential substitution with 2-amino-4-cyano-

pyridine and 3,3-diﬂuoropyrrolidine provided 23 and 24. The

tertiary alcohol 25 was synthesized as shown in Scheme 7.

Methyl Grignard addition to methyl 2,6-dichloroisonicotinate

(66) gave 2-(2,6-dichloropyridin-4-yl)propan-2-ol (67). S_NAr

displacement of the resultant 2,6-dichloropyridine 67 with 3,3-

diﬂuoropyrrolidine provided 2-amino-6-chloropyridine 68

Scheme 1. Preparation of 2-Methylpyrimidine Analogs

a

Reagents and conditions: (a) (i) R-NH₂, Pd₂(dba)₃, Xantphos, t-

BuONa, 1,4-dioxane, 80 °C, 3 h; (ii) HCl, 1,4-dioxane, MeOH, 23 °C,

16 h; (iii) benzoic acid, HBTU, Et₃N, DMF, 40 °C, 16 h; (b) (i) 4-

(triﬂuoromethyl)pyridin-2-amine, Pd₂(dba)₃, Xantphos, t-BuONa, 1,4-

dioxane, 80 °C, 3 h; (ii) HCl, 1,4-dioxane, MeOH, 23 °C, 16 h, 10%

yield.

upon treatment with phosphoryl trichloride resulted in the

( )-tert-butyl 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-car-

boxylate (38). Buchwald−Hartwig reaction of the requisite

C6-substituted-2,4-dichloropyrimidines 38−40 with 2-amino-4-

triﬂuoromethylpyridine followed by S_NAr with the selected

secondary amine and acid-mediated Boc group removal (if

appropriate) furnished 13−16, 41, and 42. Acetamide analogs

17−19 were obtained by acylation with acetic anhydride.

G

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a

Scheme 2. Preparation of 3-Piperidine, 4-Piperidine, and 4-Tetrahydropyran Analogs 13−19

a

Reagents and conditions: (a) potassium monoethyl malonate, CDI, MgCl₂, CH₃CN, 23 °C, 16 h, 86% yield; (b) urea, NaOMe, EtOH, 80 °C, 72 h,

42% yield; (c) (i) POCl₃, reﬂux, 3 h; (ii) (Boc)₂O, NaHCO₃, H₂O, THF, 23 °C, 16 h, 17% yield; (d) (i) 4-(triﬂuoromethyl)pyridin-2-amine,

Pd₂(dba)₃, Xantphos, t-BuONa, 1,4-dioxane, 80 °C, 3 h; (ii) R-amine, (i-Pr)₂NEt, DMF, 85 °C, 16 h; (iii) HCl, 1,4-dioxane, MeOH, 16 h; (e) Ac₂O,

DMAP, (i-Pr)₂NEt, CH₃CN, CH₂Cl₂, 0 °C, 1 h.

a

Scheme 3. Preparation of 20

a

Reagents and conditions: (a) Pd(dppf)Cl₂, K₂CO₃, 1,4-dioxane, H₂O, 90 °C, 56% yield; (b) 3,3-diﬂuoropyrrolidine hydrochloride, (i-Pr)₂NEt, 1,4-

dioxane, 120 °C microwave, 37% yield; (c) 2-amino-4-triﬂuoromethylpyridine, t-BuONa, RuPhos, RuPhos palladium(II) phenethylamine chloride,

120 °C microwave, 38% yield; (d) H₂, MeOH, 10% Pd/C, 40 bar, 84% crude yield. (e) HCl in 1,4-dioxane, CH₂Cl₂; (f) (i-Pr)₂NEt, THF, acetyl

chloride, 45% yield.

which upon treatment with 2-amino-4-cyanopyridine aﬀorded

25.

low tPSA and hydrogen bond donor count led to compound

26, which represents the ﬁrst potent, selective, and brain-

penetrant inhibitor of DLK reported. DLK inhibitor 26

protected primary neurons in an in vitro axon degeneration

assay as well as reduced phosphorylation of the downstream

transcription factor c-Jun, a marker of neuronal injury, in both

the optic nerve crush and MPTP mouse models of neuro-

degeneration following oral dosing.

CONCLUSIONS

■

Recent data from multiple models have revealed a central role

for dual leucine zipper kinase (DLK, MAP3K12) in the

regulation of neuronal degeneration. From a high-throughput

screen, we identiﬁed aminothiazole 1 which served as a practical

starting point for the lead optimization of a brain-penetrant

kinase inhibitor. Modiﬁcation of the 2-aminothiazole hinge

binder, pyrimidine scaﬀold, and substituents while maintaining

H

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a

Scheme 4. Preparation of 2-(4-(piperidin-4-yl)pyridin-2-

ylamino)isonicotinonitriles

Scheme 5. Preparation of 22 and 27

a

Reagents and conditions: (a) (i) 4-bromotetrahydro-2H-pyran, Zn,

TMSCl, 1,2-dibromoethane; (ii) Pd(dppf)Cl₂, CuI, DMA, 80 °C, 70%

yield; (b) 3,3-diﬂuoropyrrolidine hydrochloride, NMP, 130 °C; (c) 2-

amino-4-cyanopyridine, t-BuONa, RuPhos, RuPhos palladium(II)

phenethylamine chloride, 120 °C microwave, 29% yield; (d) (i) 1-

Boc-3-iodoazetidine, Zn, TMSCl, 1,2-dibromoethane; (ii) Pd(dppf)-

Cl₂, CuI, DMA, 80 °C, 54% yield; (e) (i) TFA, CH₂Cl₂, (ii) Et₃N, 3-

oxetanone, Na(OAc)₃BH, THF, 50 °C, 80% yield; (f) 2-amino-4-

cyanopyridine, Pd₂dba₃, Xantphos, Cs₂CO₃, 1,4-dioxane, 80 °C, 51%

yield; (g) RuPhos palladium(II) phenethylamine chloride, RuPhos,

3,3-diﬂuoropyrrolidine hydrochloride, t-BuONa, 3 Å molecular sieves,

THF, 90 °C, 25% yield.

a

Reagents and conditions: (a) (i) Zn, TMSCl, 1,2-dibromoethane; (ii)

Pd(dppf)Cl₂, CuI, DMA, 80 °C, 62% yield; (b) 3,3-diﬂuoropyrrolidine

hydrochloride, NMP, 130 °C, 47% yield; (c) (i) TFA, CH₂Cl₂, (ii)

Et₃N, 3-oxetanone, Na(OAc)₃BH, THF; 85% yield for 53, 99% crude

yield for 54a; (d) 2-amino-4-cyanopyridine, Pd₂(dba)₃, BINAP,

Cs₂CO₃, 1,4-dioxane, reﬂux, 77% yield; (e) (i) TFA, CH₂Cl₂, (ii)

Ac₂O, Et₃N, DMAP, CH₂Cl₂; quantitative (crude); (f) 2-amino-4-

cyanopyridine, Pd₂(dba)₃, Xantphos, Cs₂CO₃, 1,4-dioxane, 80 °C; 78%

yield; (g) RuPhos palladium(II) phenethylamine chloride, RuPhos, R-

amine, t-BuONa, 3 Å molecular sieves, THF, 90 °C; (h) R−OH,

Pd(OAc)₂, Ad-BippyPhos, Cs₂CO₃, 3 Å molecular sieves, toluene,

H₂O, 120 °C; (i) Pd(OAc)₂, n-BuPAd₂, R-BF₃K, Cs₂CO₃, toluene,

H₂O, 110 °C.

X-100, blocked for 1 h with SuperBlock before the overnight

incubation with the primary antibodies at 4 °C. The secondary

antibodies were incubated for 2 h, washed with PBS, and then stained

with Hoechst 33342 dye. The cell plates were imaged on Opera

Imaging Platform.

In Vitro Axon Degeneration Cell Assay. Assay was conducted as

previous described^14,64with the following modiﬁcations. Dorsal root

ganglion (DRG) neurons were freshly dissected from E14.5 rat

embryos. The resulting cell suspension was ﬁltered through a 50 μm

sieve (Partec) to remove remaining tissue pieces, centrifuged 5 min at

1000 rpm, and resuspended in DRG culture medium (DMEM/F12

containing 1× N3 supplement, 0.18% glucose, 25 ng/mL NGF).

Neurons were then plated on a 384-well dish at a density of 1200−

2000 cells per well on top of the astrocyte monolayer. To inhibit cell

proliferation, medium was supplied with 200 μM uridine and 100 μM

5-ﬂuorodeoxyuridine the next day. DRGs were cultured for 4 days

prior to the assay.

EXPERIMENTAL SECTION

Methods. DLK Biochemical Assay. The biochemical assay was

■

performed as previously described.²²

p-JNK Cell Assay. 7500 HEK293 cells stably transfected with Dox-

inducible human DLK in 40 μL of DMEM with 10% serum were

seeded into each well of 384-well poly-^D-lysine coated plates. The

plates were incubated at 37 °C for 20−24 h prior to the addition of 5

μL of 60 μM doxycycline in DMEM. After incubation with doxycycline

at 37 °C for approximately 20 h, 5 μL of DLK inhibitors in DMEM

was added, and cells were incubated at 37 °C for an additional 5.5 h.

The cells were then washed with PBS, permeabilized with 0.1% Triton

In Vitro Transporter Assays. Madin−Darby kidney cells

(MDCK) stably transfected with human MDR1 (Pgp) were obtained

from the National Institutes of Health, (Bethesda, MD). Cells were

I

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a

harvested with trypsin and seeded on Millipore Millicell 24-well plates

at initial concentrations of 2.0 × 10⁵cells/mL and allowed to grow for

5 days. Cell monolayers were equilibrated in transport buﬀer (Hank’s

balanced salt solution with 10 mM Hepes, pH 7.4) for 60 min at 37 °C

with 5% CO₂and 95% relative humidity prior to the experiment. Dose

solutions were prepared in transport buﬀer and consisted of test

compounds (5 μM) and the monolayer integrity marker lucifer yellow

(100 μM). The dose solutions were added to the donor chambers, and

transport buﬀer was added to all receiver chambers. The transport was

examined in the apical to basolateral (A−B) and basolateral to apical

(B−A) directions. The receiver chambers were sampled (50 μL) at 60,

120, and 180 min and were replenished with fresh transport buﬀer

after the 60 and 120 min samplings. Lucifer yellow permeability was

used as a marker of monolayer integrity during the experiment.

Compound concentrations in the donor and receiving compartments

were determined by LC−MS/MS analysis. The apparent permeability

(P_app) in the apical to basal A−B and basal to apical B−A directions

was calculated as follows: P_app= (dQ/dt)[1/(AC₀)], where dQ/dt =

rate of compound appearance in the receiver compartment, A =

surface area of the inset, and C₀= initial substrate concentration at T =

0. The eﬄux ratio (ER) was calculated as P_app(B−A)/P_app(A−B).

Animal Models. All experiments with mice were performed under

animal protocols approved by the Animal Care and Use Committee at

Genentech and adhere to ACS Ethical Guidelines for animal studies.

For all in vivo studies, C57Bl/6 mice were dosed with compound 26

orally as an MCT suspension. Optic nerve crush studies were

conducted as described,¹⁷except p-c-Jun was measured at 6 h by MSD

ELISA (Meso Scale Discovery). For MPTP studies, animals were

administered four ip doses of 20 mg/kg MPTP (1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine), with each dose separated by 4 h. Twenty-

ﬁve hours after the ﬁrst MPTP dose, animals were sacriﬁced and

processed essentially as described.¹¹For each animal, the p-c-Jun

positive cell number/mm²in ﬁve sections was averaged to generate a

value for that animal.

Scheme 6. Preparation of Tertiary Nitriles 23 and 24

Synthesis. General. All commercially available reagents and

solvents were used as received. Reactions using air- or moisture-

sensitive reagents were performed under an atmosphere of nitrogen

using freshly opened EMD DriSolv solvents. Reaction progress was

monitored by TLC and/or LCMS. Flash column chromatography was

performed with Isco CombiFlash Companion systems using prepacked

silica gel columns (40−60 μm particle size RediSep or 20−40 μm

spherical silica gel RediSep Gold columns or similar columns from

other vendors). Preparative reverse phase HPLC puriﬁcations were

performed on a Varian Prostar instrument, using a Phenomenex

Gemini-NX C-18 (3 cm × 5 cm, 5 μm) stationary phase, with 0.1%

aqueous formic acid/acetonitrile or 0.1% aqueous ammonium

hydroxide/acetonitrile gradients as the mobile phase (typically 5−

85% acetonitrile over 10 min) with a ﬂow rate of 60 mL/min. NMR

spectra were measured on a Bruker 300 or 400 MHz spectrometer,

and chemical shifts were reported in ppm downﬁeld from TMS using

residual nondeuterated solvent as internal standards (CHCl₃, 7.26

ppm; DMSO, 2.50 ppm; MeOH, 3.31 ppm). The following

abbreviations are used: br = broad signal, s = singlet, d = doublet,

dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. The

purity of ﬁnal compounds was veriﬁed by HPLC to be >95% in all

cases using either of the following methods: (1) Agilent 1200

instrument with an Agilent SB C-18 (2.1 mm × 30 mm, 1.8 μm

particle size) stationary phase and a gradient of water/acetonitrile (5−

95% over 10 min, 0.05% TFA in both phases) at a ﬂow rate of 0.4 mL/

min. Quantiﬁcation of target and impurities was done by UV detection

at 254 nm. (2) Shimadzu LC-2010A/2020A instrument with an

Ultimate C-18 (3.0 mm × 50 mm, 3 μm particle size) stationary phase

and a gradient of water/acetonitrile (10−80% over 6 min and then

80% over 2 min, 0.05% TFA in both phases) at a ﬂow rate of 1.2 mL/

min and oven temperature of 40 °C. Quantiﬁcation of target and

impurities was done by UV detection at 254 nm.

a

Reagents and conditions: (a) tetrahydro-2H-pyran-4-carbonitrile,

LHMDS, THF, −78 to 24 °C, 71% yield; (b) (CH₃)₂CHCN,

LHMDS, THF, −78 to 24 °C, 84% yield; (c) 2-amino-4-

cyanopyridine, Pd₂(dba)₃, BINAP, Cs₂CO₃, 1,4-dioxane, 80 °C, 44%

yield; (d) 3,3-diﬂuoropyrrolidine hydrochloride, (i-Pr)₂NEt, DMSO,

80−90 °C, 50% yield; (e) RuPhos palladium(II) phenethylamine

chloride, RuPhos, 2-amino-4-cyanopyridine, t-BuONa, 3 Å molecular

sieves, THF, 90 °C, 47% yield; (f) 2-amino-4-cyanopyridine, Pd(P(t-

Bu)₃)₂, K₃PO₄, 1,4-dioxane, reﬂux, 4% yield.

a

Scheme 7. Preparation of Tertiary Alcohol 25

a

Reagents and conditions: (a) CH₃MgBr, THF, −78 °C, 70% yield;

(b) 3,3-diﬂuoropyrrolidine hydrochloride, (i-Pr)₂NEt, DMSO, 80−90

°C, 50% yield; (c) 2-amino-4-cyanopyridine, Pd(P(t-Bu)₃)₂, K₃PO₄,

1,4-dioxane, reﬂux. 62% yield.

( )-(3-(2-Methyl-6-((5-methylthiazol-2-yl)amino)pyrimidin-

4-yl)piperidin-1-yl)(phenyl)methanone (1). A mixture of ( )-tert-

butyl 3-(6-chloro-2-methylpyrimidin-4-yl)piperidine-1-carboxylate

(34) (62 mg, 0.20 mmol), 5-methylthiazol-2-amine (32 mg, 0.28

maintained in Dulbecco’s modiﬁed Eagle medium supplemented with

10% FBS, 80 ng/mL colchicine, and 5 μg/mL Plasmocin. Cells were

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mmol), sodium tert-butoxide (27 mg, 0.28 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (35 mg, 0.060 mmol),

and tris(dibenzylideneacetone)dipalladium(0) (55 mg, 0.060 mmol)

in 1,4-dioxane (10 mL) was stirred at 90 °C for 16 h. The reaction

mixture was ﬁltered and diluted with water (15 mL). The resulting

solution was extracted with ethyl acetate (2 × 15 mL). The collected

organic was dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. The crude product was

dissolved in methanol (2 mL), and 4.0 M solution of hydrogen

chloride in 1,4-dioxane (0.5 mL, 2 mmol) was added. After 16 h, the

mixture was concentrated under reduced pressure. The resulting crude

amine, benzoic acid (29 mg, 0.24 mmol), triethylamine (0.139 mL,

0.997 mmol), and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-

uronium hexaﬂuorophosphate (114 mg, 0.301 mmol) in N,N-

dimethylformamide were stirred at 23 °C for 16 h. The mixture was

concentrated under reduced pressure and puriﬁed by preparative

reverse phase HPLC to aﬀord the title compound as an oﬀ-white solid

( )-(3-(6-((4-Cyclopropylpyridin-2-yl)amino)-2-methylpyri-

midin-4-yl)piperidin-1-yl)(phenyl)methanone (8). The title

compound (28 mg, 34% yield) was prepared in a manner analogous

to 1 by substituting 4-cyclopropylpyridin-2-amine for 5-methylthiazol-

2-amine. LCMS: m/z = 414.2 [M + H]⁺. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.03−9.80 (m, 1H), 8.10 (d, J = 5.3 Hz, 1H), 7.70−7.19

(m, 7H), 6.79−6.50 (m, 1H), 4.87−4.21 (m, 1H), 3.81−3.47 (m, 1H),

3.21−2.79 (m, 2H), 2.83−2.66 (m, 2H), 2.09−1.43 (m, 7H), 1.13−

0.95 (m, 2H), 0.84−0.44 (m, 2H).

(

)-(3-(2-Methyl-6-(pyridin-2-ylamino)pyrimidin-4-yl)-

piperidin-1-yl)(phenyl)methanone (9). The title compound (16

mg, 21% yield) was prepared in a manner analogous to 1 by

substituting pyridin-2-amine for 5-methylthiazol-2-amine. LCMS: m/z

1

= 374.1 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ 10.07 (s, 1H),

8.59−8.38 (m, 1H), 8.26−7.95 (m, 1H), 7.64−7.28 (m, 5H), 7.06−

6.88 (m, 1H), 4.72−4.28 (m, 1H), 3.86−3.42 (m, 1H), 3.21−2.89 (m,

3H), 2.87−2.57 (m, 1H), 2.22−1.93 (m, 1H), 1.95−1.37 (m, 2H).

( )-2-((6-(1-Benzoylpiperidin-3-yl)-2-methylpyrimidin-4-yl)-

amino)isonicotinonitrile (10). The title compound (37 mg, 46%

yield) was prepared in a manner analogous to 1 by substituting 2-

aminoisonicotinonitrile for 5-methylthiazol-2-amine. LCMS: m/z =

1

(16 mg, 21% yield). LCMS: m/z = 394.2 [M + H]⁺. H NMR (400

MHz, DMSO-d₆) δ 11.36 (s, 1H), 7.62−7.27 (m, 5H), 7.09 (s, 1H),

6.73 (s, 1H), 4.86−4.25 (m, 1H), 3.95−3.52 (m, 1H), 3.16−2.64 (m,

3H), 2.35 (s, 3H), 2.11−1.90 (m, 1H), 1.90−1.32 (m, 3H).

1

399.2 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H),

( )-(3-(2-Methyl-6-((5-methylpyridin-2-yl)amino)pyrimidin-

4-yl)piperidin-1-yl)(phenyl)methanone (2). The title compound

(49 mg, 63% yield) was prepared in a manner analogous to 1 by

substituting 5-methylpyridin-2-amine for 5-methylthiazol-2-amine.

8.52 (d, J = 5.2 Hz, 1H), 8.12 (s, 1H), 7.47−7.27 (m,7H), 4.63−4.38

(m, 1H), 3.76−3.55 (m, 1H), 3.14−2.63 (m, 3H), 2.56−2.39 (m, 1H),

2.10−1.97 (m, 1H), 1.88−1.47 (m, 3H).

1

LCMS: m/z = 388.3 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ

(

)-(3-(6-((4-Methoxypyridin-2-yl)amino)-2-methylpyrimi-

din-4-yl)piperidin-1-yl)(phenyl)methanone (11). The title com-

pound (37 mg, 45% yield) was prepared in a manner analogous to 1

by substituting for 4-methoxypyridin-2-amine for 5-methylthiazol-2-

amine. LCMS: m/z = 404.2 [M + H]⁺. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.01 (s, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.55−7.27 (m, 7H),

6.65−6.57 (m, 1H), 4.64−4.40 (m, 1H), 3.81 (s, 3H), 3.72−3.55 (m,

1H), 3.15−2.89 (m, 2H), 2.80−2.67 (m, 1H), 2.50−2.31 (m, 2H),

2.04−1.44 (m, 4H).

9.94 (s, 1H), 8.12 (s, 1H), 7.65−7.32 (m, 8H), 4.68−4.37 (m, 1H),

3.82−3.49 (m, 1H), 3.16−2.84 (m, 1H), 2.75−2.61 (m, 1H), 2.48−

2.12 (m, 6H), 2.12−1.93 (m, 1H), 1.89−1.46 (m, 3H).

( )-(3-(2-Methyl-6-((4-methylpyridin-2-yl)amino)pyrimidin-

4-yl)piperidin-1-yl)(phenyl)methanone (3). The title compound

(25 mg, 32% yield) was prepared in a manner analogous to 1 by

substituting 4-methylpyridin-2-amine for 5-methylthiazol-2-amine.

1

LCMS: m/z = 388.1 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ

(

)-2-Methyl-6-(piperidin-3-yl)-N-(4-(triﬂuoromethyl)-

9.97 (s, 1H), 8.14 (d, J = 5.1 Hz, 1H), 7.74−7.31 (m, 7H), 6.94−6.73

(m, 1H), 4.74−4.35 (m, 1H), 3.86−3.45 (m, 1H), 3.19−2.62 (m, 3H),

2.42−2.21 (m, 4H), 2.13−1.92 (m, 1H), 1.90−1.42 (m, 3H).

( )-(3-(2-Methyl-6-((5-(triﬂuoromethyl)pyridin-2-yl)amino)-

pyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (4). The title

compound (60 mg, 68% yield) was prepared in a manner analogous to

1 by substituting 5-(triﬂuoromethyl)pyridin-2-amine for 5-methyl-

thiazol-2-amine. LCMS: m/z = 442.1 [M + H]⁺. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.57 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 8.25−7.97 (m,

1H), 7.88 (s, 1H), 7.73−7.25 (m, 6H), 4.79−4.28 (m, 1H), 3.91−3.38

(m, 1H), 3.21−2.88 (m, 2H), 2.88−2.63 (m, 1H), 2.25−1.93 (m, 1H),

1.93−1.25 (m, 3H).

( )-(3-(2-Methyl-6-((4-(triﬂuoromethyl)pyridin-2-yl)amino)-

pyrimidin-4-yl)piperidin-1-yl)(phenyl)methanone (5). The title

compound (18 mg, 20% yield) was prepared in a manner analogous to

1 by substituting 4-(triﬂuoromethyl)pyridin-2-amine for 5-methyl-

thiazol-2-amine. LCMS: m/z = 442.4 [M + H]⁺. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.87 (s, 1H), 9.21 (s, 1H), 8.15−7.51 (m, 4H), 6.70−

5.79 (m, 4H), 3.69−3.44 (m, 1H), 3.16−2.93 (m, 3H), 1.63−1.43 (m,

1H), 1.37−1.03 (m, 4H), 1.03−0.76 (m, 3H).

pyridin-2-yl)pyrimidin-4-amine (12). A mixture of ( )-tert-butyl

3-(6-chloro-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (34) (62

mg, 0.20 mmol), 4-(triﬂuoromethyl)pyridin-2-amine (45 mg, 0.28

mmol), sodium tert-butoxide (27 mg, 0.28 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (35 mg, 0.060 mmol),

and tris(dibenzylideneacetone)dipalladium(0) (55 mg, 0.060 mmol)

in 1,4-dioxane (10 mL) was stirred at 90 °C for 16 h. The reaction

mixture was ﬁltered and diluted with water (15 mL). The resulting

solution was extracted with ethyl acetate (2 × 15 mL). The collected

organic was dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. The crude product was

dissolved in methanol (2 mL), and 4.0 M solution of hydrogen

chloride in 1,4-dioxane (0.5 mL, 2 mmol) was added to the solution.

After 16 h, the mixture was concentrated under reduced pressure and

puriﬁed by preparative reverse phase HPLC to aﬀord the title

compound as an oﬀ-white solid (7 mg, 10% yield). LCMS: m/z =

1

388.1 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ 10.44 (s, 1H),

8.55 (d, J = 5.2 Hz, 1H), 8.19 (s, 1H), 7.40 (s, 1H), 7.36−7.22 (m,

1H), 3.17 (s, 2H), 3.05 (d, J = 7.8 Hz, 1H), 2.97−2.88 (m, 1H), 2.76−

2.57 (m, 2H), 1.99−1.87 (m, 1H), 1.80 (s, 3H), 1.73−1.55 (m, 2H),

1.54−1.40 (m, 1H).

( )-(3-(6-((4-Ethylpyridin-2-yl)amino)-2-methylpyrimidin-4-

yl)piperidin-1-yl)(phenyl)methanone (6). The title compound (36

mg, 41% yield) was prepared in a manner analogous to 1 by

substituting 4-ethylpyridin-2-amine for 5-methylthiazol-2-amine.

( )-tert-Butyl 3-(3-Ethoxy-3-oxopropanoyl)piperidine-1-car-

boxylate (36). To a solution of piperidine-1,3-dicarboxylic acid 1-tert-

butyl ester (35) (160 g, 0.70 mol) in acetonitrile (1.6 L) was added

CDI (136 g, 0.839 mol) portionwise at 23 °C. After 1 h, potassium

monoethylmalonate (119 g, 0.697 mol) and MgCl₂(66.4 g, 0.697

mol) were added. After 16 h, the reaction mixture was concentrated

under reduced pressure. The residue was diluted with cold water, and

the resulting solution was neutralized with citric acid. The mixture was

extracted with dichloromethane (3 × 600 mL). The collected organic

was concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography (2:1 heptane/ethyl acetate) aﬀorded the title

1

LCMS: m/z = 442.4 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ

9.97 (s, 1H), 8.46−8.24 (m, 1H), 8.26−8.02 (m, 1H), 7.80−7.35 (m,

6H), 6.97−6.78 (m, 1H), 2.83−2.53 (m, 5H), 2.10−1.89 (m, 2H),

1.96−1.48 (m, 4H), 1.33−1.04 (m, 4H).

( )-(3-(6-((4-Isopropylpyridin-2-yl)amino)-2-methylpyrimi-

din-4-yl)piperidin-1-yl)(phenyl)methanone (7). The title com-

pound (31 mg, 37% yield) was prepared in a manner analogous to 1

by substituting 4-isopropylpyridin-2-amine for 5-methylthiazol-2-

amine. LCMS: m/z = 416.3 [M + H]⁺. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.01 (s, 1H), 8.32−8.05 (m, 2H), 7.75−7.23 (m, 6H),

6.97−6.76 (m, 1H), 4.79−4.30 (m, 2H), 3.78−3.48 (m, 1H), 3.11−

2.62 (m, 6H), 2.44−2.21 (m, 2H), 2.13−1.46 (m, 7H).

1

compound as an oﬀ-white solid (180 g, 86%). H NMR (400 MHz,

DMSO-d₆at 80 °C) δ 4.14−3.91 (m, 2H), 3.87−3.69 (m, 1H), 3.64−

3.00 (m, 3H), 2.90−2.88 (m, 1H), 2.63−2.50 (m, 1H), 2.63−2.50 (m,

K

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1H), 2.50−2.48 (m, 1H), 1.90 (m, 1H), 1.64−1.63 (m, 1H), 1.53−

1.40 (m, 1H), 1.35 (s, 9H), 1.28 (m, 1H), 1.2 (m, 3H).

2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(piperidin-4-yl)-N-(4-

(triﬂuoromethyl)pyridin-2-yl)pyrimidin-4-amine (15). The title

compound (32 mg, 74% yield) was prepared in a manner analogous to

13 by substituting tert-butyl 4-(2,6-dichloropyrimidin-4-yl)piperidine-

1-carboxylate (39) and 3,3-diﬂuoropyrrolidine hydrochloride for tert-

butyl 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38) and

tert-Butyl 3-(2,6-Dihydroxypyrimidin-4-yl)piperidine-1-car-

boxylate (37). A solution of ( )-tert-butyl 3-(3-ethoxy-3-

oxopropanoyl)piperidine-1-carboxylate (36) (180 g, 0.60 mol), urea

(361 g, 6.02 mol), and sodium methoxide (325 g, 6.02 mol) in ethanol

(1.8 L) was reﬂuxed for 72 h. After completion of the reaction, the

solvent was concentrated under reduced pressure. The residue was

dissolved in water and neutralized with citric acid. The resulting

solution was extracted with dichloromethane (3 × 800 mL). The

collected organic was sequentially washed with water (2 × 800 mL),

saturated aqueous sodium chloride solution (800 mL) and

concentrated under reduced pressure. Trituration with petroleum

ether aﬀorded the title compound as a white solid (75 g, 42%). LCMS:

m/z = 294.6 [M − H]⁻. ¹H NMR (300 MHz, DMSO-d₆) δ 10.96 (br

s, 1H), 10.81 (br s, 1H), 5.3 (s, 1H), 3.79−3.75 (m, 2H), 2.82−2.75

(m, 2H), 2.34 (m, 1H), 1.89−1.83 (m, 1H), 1.58−1.54 (m, 2H), 1.37

(m, 10H).

1

pyrrolidine, respectively. LCMS: m/z = 429.2 [M + H]⁺. H NMR

(400 MHz, DMSO-d₆) δ 10.29 (s, 1H), 8.71 (s, 1H), 8.52 (d, J = 5.1

Hz, 1H), 7.29 (d, J = 5.1 Hz, 1H), 6.48 (s, 1H), 3.89 (t, J = 13.2 Hz,

2H), 3.73 (t, J = 7.3 Hz, 2H), 3.06 (d, J = 12.1 Hz, 2H), 2.67−2.52 (m,

4H), 1.92−1.68 (m, 2H), 1.58 (m, 2H).

2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(tetrahydro-2H-pyran-4-yl)-

N-(4-(triﬂuoromethyl)pyridin-2-yl)pyrimidin-4-amine (16). The

title compound (31 mg, 72% yield) was prepared in a manner

analogous to 13 by substituting 2,4-dichloro-6-(tetrahydro-2H-pyran-

4-yl)pyrimidine (40) and 3,3-diﬂuoropyrrolidine hydrochloride for

tert-butyl 3-(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38)

and pyrrolidine, respectively. LCMS: m/z = 430.2 [M + H]⁺. ¹H NMR

(400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 8.70 (s, 1H), 8.52 (d, J = 5.2

Hz, 1H), 7.28 (d, J = 4.9 Hz, 1H), 6.50 (s, 1H), 3.97−3.84 (m, 4H),

3.79−3.60 (m, 2H), 3.43 (td, J = 11.1, 3.5 Hz, 2H), 2.72−2.54 (m,

2H), 1.90- 1.59 (m, 5H).

tert-Butyl 3-(2,6-Dichloropyrimidin-4-yl)piperidine-1-car-

boxylate (38). tert-Butyl 3-(2,6-dihydroxypyrimidin-4-yl)piperidine-

1-carboxylate (37) (75 g, 0.25 mol) was dissolved in phosphoryl

trichloride (750 mL) and heated to reﬂux for 3 h. The mixture was

concentrated to 10% of the original volume and poured slowly onto

ice. The solution was adjusted to pH ≈ 9 with slow addition of solid

sodium bicarbonate. Tetrahydrofuran (500 mL) and di-tert-butyl

dicarbonate (83 g, 0.38 mol) were added, and the resulting mixture

was stirred at 23 °C for 16 h. The mixture was ﬁltered, and the ﬁltrate

was extracted with dichloromethane (3 × 400 mL). The collected

organic was washed sequentially with water (3 × 400 mL), saturated

aqueous sodium chloride solution (400 mL) and concentrated under

reduced pressure. Puriﬁcation by ﬂash column chromatography (1:1

heptane/ethyl acetate) aﬀorded the title compound as a white solid

1-(4-(2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(4-(triﬂuoromethyl)-

pyridin-2-ylamino)pyrimidin-4-yl)piperidin-1-yl)ethanone

(17). To an ice-cooled solution of 2-(3,3-diﬂuoropyrrolidin-1-yl)-6-

(piperidin-4-yl)-N-(4-(triﬂuoromethyl)pyridin-2-yl)pyrimidin-4-amine

(15) (42 mg, 0.10 mmol), 4-(dimethylamino)pyridine (1.2 mg, 0.0098

mmol) and N,N-diisopropylethylamine (65 mg, 0.50 mmol) in

acetonitrile (5 mL) and dichloromethane (5 mL) was added acetic

anhydride (0.020 mL, 0.22 mmol). After 1 h, the reaction mixture was

poured into saturated aqueous ammonium chloride solution (10 mL),

and the resulting solution was extracted with ethyl acetate (3 × 5 mL).

The combined organic was washed saturated aqueous sodium chloride

solution (10 mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. Puriﬁcation by preparative

reverse phase HPLC aﬀorded the title compound as an oﬀ-white solid

t

1

(14 g, 17%). LCMS: m/z = 278 [M − Bu + 2H]⁺. H NMR (400

MHz, DMSO-d₆) δ 7.79 (s, 1H), 4.00 (br s, 1H), 3.80 (br s, 1H),

3.20−3.00 (m, 1H), 2.90−2.84 (m, 2H), 1.95 (m, 1H), 1.72−1.60 (m,

2H), 1.38 (m, 10H).

1

(37 mg, 80% yield). LCMS: m/z = 471.2 [M + H]⁺. H NMR (400

6-(Piperidin-3-yl)-2-(pyrrolidin-1-yl)-N-(4-(triﬂuoromethyl)-

pyridin-2-yl)pyrimidin-4-amine (13). A solution of tert-butyl 3-

(2,6-dichloropyrimidin-4-yl)piperidine-1-carboxylate (38) (33 mg,

0.10 mmol), 4-(triﬂuoromethyl)pyridin-2-amine (23 mg, 0.14

mmol), sodium tert-butoxide (14 mg, 0.14 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (18 mg, 0.031 mmol),

and tris(dibenzylideneacetone)dipalladium(0) (28 mg, 0.030 mmol)

in 1,4-dioxane (5 mL) was stirred at 23 °C for 1 h before heating to 60

°C for 13 h. The mixture was ﬁltered and diluted with water (10 mL).

The resulting solution was extracted with ethyl acetate (2 × 10 mL).

The collected organic was dried over anhydrous sodium sulfate,

ﬁltered, and concentrated under reduced pressure. The residue was

dissolved in N,N-dimethylformamide (5 mL) before the addition of

pyrrolidine (21 mg, 0.30 mmol) and N,N-diisopropylethylamine (64

mg, 0.50 mmol). The reaction mixture was heated to 85 °C. After 16

h, the reaction was concentrated under reduced pressure. The resulting

residue was dissolved in methanol (2 mL) before the addition of a 4.0

M solution of hydrogen chloride in 1,4-dioxane (0.5 mL, 2 mmol).

After 16 h, the mixture was concentrate under reduced pressure.

Puriﬁcation by preparative reverse phase HPLC aﬀorded the title

compound as an oﬀ-white solid (14 mg, 37% yield). LCMS: m/z =

MHz, DMSO-d₆) δ 10.31 (s, 1H), 8.70 (s, 1H), 8.52 (d, J = 5.2 Hz,

1H), 7.31−6.90 (m, 1H), 6.49 (s, 1H), 4.63−4.35 (m, 1H), 4.02−3.84

(m, 3H), 3.73 (t, J = 7.3 Hz, 2H), 3.25−3.02 (m, 1H), 2.74−2.55 (m,

4H), 1.83 (t, J = 14.5 Hz, 2H), 1.70−1.37 (m, 2H).

1-(4-(2-(3,3-Diﬂuoroazetidin-1-yl)-6-((4-(triﬂuoromethyl)-

pyridin-2-yl)amino)pyrimidin-4-yl)piperidin-1-yl)ethanone

(18). The title compound (32 mg, 68% yield) was prepared in a

manner analogous to 17 by substituting 3,3-diﬂuoroazetidine hydro-

chloride for 3,3-diﬂuoropyrrolidine hydrochloride. LCMS: m/z =

1

457.1 [M + H]⁺. H NMR (400 MHz, CD₃OD) δ 8.53 (s, 1H), 8.46

(d, J = 5.6 Hz, 1 H), 7.21 (d, J = 4.8 Hz, 1 H), 6.60 (s, 1H), 4.65−4.61

(m, 1 H), 4.44 (t, J = 12.0 Hz, 4 H), 4.05−4.01 (m, 1 H), 3.29−3.19

(m, 1 H), 2.79−2.69 (m, 2H), 2.12 (s, 3 H), 2.00−1.91 (m, 2H),

1.75−1.66 (m, 2 H).

1-(4-(2-Morpholino-6-((4-(triﬂuoromethyl)pyridin-2-yl)-

amino)pyrimidin-4-yl)piperidin-1-yl)ethanone (19). The title

compound (34 mg, 75% yield) was prepared in a manner analogous

to 17 by substituting morpholine for 3,3-diﬂuoropyrrolidine hydro-

chloride. LCMS: m/z = 451.0 [M + H]⁺. ¹H NMR (400 MHz,

CDCl₃), δ ppm: 8.54−8.43 (m, 2 H), 7.31−7.26 (m, 2H), 6.90−6.60

(br s, 1H), 4.90−4.60 (m, 1H), 4.18−3.87 (m, 8H), 3.60−3.40 (m,

1H), 2.80−2.70 (m, 1H), 2.52−2.50 (m, 1H), 2.30−2.26 (m, 4H),

2.10−1.96 (m, 1H), 1.80−1.40 (m, 3H).

tert-Butyl 4-(2,6-Dichloropyridin-4-yl)-5,6-dihydropyridine-

1(2H)-carboxylate (45). A mixture of 2,6-dichloro-4-iodopyridine

(43) (1.0 g, 3.7 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-5,6-dihydro-2H-pyridine-1-carboxylate (44) (1.4

g, 4.4 mmol), potassium carbonate (1.0 g, 7.3 mmol), and

Pd(dppf)Cl₂·CH₂Cl₂(0.30 g, 0.37 mmol) in 1,4-dioxane (12 mL)

and water (3 mL) was purged with nitrogen and heated at 90 °C for

16 h. The mixture was diluted with water (100 mL), and the resulting

mixture was extracted with ethyl acetate (2 × 100 mL). The combined

organic was washed with saturated aqueous sodium chloride solution,

dried over magnesium sulfate, ﬁltered, and concentrated under

1

393.2 [M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ 10.18 (s, 1H),

8.85 (s, 1H), 8.50 (d, J = 5.1 Hz, 1H), 7.26 (d, J = 4.7 Hz, 1H), 6.32

(s, 1H), 3.19 (d, J = 12.4 Hz, 2H), 3.04 (s, 2H), 2.78 (t, J = 11.5 Hz,

1H), 2.64 (dd, J = 15.1, 6.7 Hz, 2H), 1.94 (s, 5H), 1.80−1.46 (m, 4H).

2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(piperidin-3-yl)-N-(4-

(triﬂuoromethyl)pyridin-2-yl)pyrimidin-4-amine (14). The title

compound (18 mg, 43% yield) was prepared in a manner analogous to

13 by substituting 3,3-diﬂuoropyrrolidine hydrochloride for pyrroli-

dine. LCMS: m/z = 429.2 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.29 (d, J = 17.8 Hz, 1H), 8.69 (s, 1H), 8.53 (d, J = 5.1 Hz, 1H),

7.29 (d, J = 5.1 Hz, 1H), 6.48 (s, 1H), 3.90 (t, J = 13.2 Hz, 3H), 3.73

(t, J = 7.3 Hz, 3H), 3.03 (d, J = 12.6 Hz, 2H), 2.82−2.56 (m, 4H), 1.92

(d, J = 13.7 Hz, 1H), 1.77−1.50 (m, 3H).

L

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reduced pressure. Puriﬁcation by ﬂash column chromatography (15%

ethyl acetate in heptane) aﬀorded the title compound as a light yellow

aﬀorded the title product as a yellow solid (25 mg, 45% yield). LCMS:

m/z = 470 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.82 (s, 1H),

8.67 (s, 1H), 8.43 (d, J = 5.1 Hz, 1H), 7.13 (d, J = 5.2 Hz, 1H), 6.50

(s, 1H), 5.98 (s, 1H), 4.52 (d, J = 13.2 Hz, 1H), 3.91 (d, J = 13.5 Hz,

1H), 3.83 (t, J = 13.3 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 3.12 (t, J =

12.2 Hz, 1H), 2.69−2.53 (m, 3H), 2.03 (s, 3H), 1.77 (t, J = 13.8 Hz,

2H), 1.64−1.51 (m, 1H), 1.50−1.33 (m, 2H).

1

solid (670 mg, 56% yield). LCMS: m/z = 330 [M + H]⁺. H NMR

(400 MHz, CDCl₃) δ 7.21 (s, 2H), 6.32 (s, 1H), 4.12 (d, J = 2.7 Hz,

2H), 3.63 (t, J = 5.6 Hz, 2H), 2.45 (s, 2H), 1.49 (s, 9H).

tert-Butyl 4-(2-Chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-

4-yl)-5,6-dihydropyridine-1(2H)-carboxylate (46). A mixture of

tert-butyl 4-(2,6-dichloro-4-pyridyl)-3,6-dihydro-2H-pyridine-1-carbox-

ylate (45) (240 mg, 0.72 mmol), 3,3-diﬂuoropyrrolidine hydrochloride

(126 mg, 0.878 mmol), and N,N-diisopropylethylamine (0.38 mL, 2.2

mmol) in 1,4-dioxane (3.0 mL) was heated in a CEM microwave at

120 °C. After 30 min, additional 3,3-diﬂuoropyrrolidine hydrochloride

(251 mg, 1.75 mmol) and N,N-diisopropylethylamine (0.35 mL, 2.01

mmol) in N,N-dimethylformamide (1.0 mL) were added to the

reaction mixture. The resulting solution was heated at 130 °C under

microwave irradiation for 30 min and then heated in an oil bath at 110

°C for 16 h The reaction mixture was diluted with water (50 mL) and

extracted with ethyl acetate (2 × 50 mL). The collected organic was

dried over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation by ﬂash column chromatography (9:1

heptane/ethyl acetate) aﬀorded the title compound as a thick oil (106

mg, 37% yield). LCMS: m/z = 400 [M + H]⁺.

tert-Butyl 4-(2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(4-

(triﬂuoromethyl)pyridin-2-ylamino)pyridin-4-yl)-5,6-dihydro-

pyridine-1(2H)-carboxylate (47). A mixture of tert-butyl 4-[2-

chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)-4-pyridyl]-3,6-dihydro-2H-pyri-

dine-1-carboxylate (46) (150 mg, 0.37 mmol), 4-(triﬂuoromethyl)-

pyridin-2-amine (73 mg, 0.45 mmol), sodium tert-butoxide (54 mg,

0.56 mmol), 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (22

mg, 0.047 mmol), and RuPhos palladium(II) phenethylamine chloride

(27 mg, 0.033 mmol) in 1,4-dioxane (2.0 mL) was capped in a

microwave vial, purged with nitrogen, and heated at 120 °C under

microwave irradiation (CEM) for 15 min. The reaction mixture was

diluted with ethyl acetate (15 mL), ﬁltered through Celite, and

concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography (4:1 heptane/ethyl acetate) yielded the title

compound as an oﬀ-white solid (75 mg, 38% yield). LCMS: m/z =

526 [M + H]⁺.

tert-Butyl 4-(2,6-Dichloro-4-pyridyl)piperidine-1-carboxy-

late (51). To a suspension of zinc dust (4.06 g, 62.1 mmol) in

N,N-dimethylacetamide (5 mL) under a nitrogen atmosphere was

cautiously added a mixture of trimethylsilyl chloride (0.946 mL, 7.30

mmol) and 1,2-dibromoethane (0.636 mL, 7.30 mmol) over 10 min.

After stirring for 15 min, a solution of N-(tert-butoxycarbonyl)-4-

iodopiperidine (50) (16.74 g, 51.11 mmol) in N,N-dimethylacetamide

(20 mL) was added over 30 min and stirring was continued for an

additional 30 min. In the open atmosphere, this mixture was ﬁltered

through Celite as quickly as possible, rinsing with a small amount of

N,N-dimethylacetamide. The resulting yellow solution was slowly

added to a separately prepared, nitrogen ﬂushed suspension of [1,1-

bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.35 g, 1.83

mmol), copper(I) iodide (695 mg, 3.65 mmol), and 2,6-dichloro-4-

iodopyridine (43) (10.0 g, 36.5 mmol) in N,N-dimethylacetamide (30

mL), and this mixture was stirred at 80 °C for 16.5 h. After cooling to

23 °C, the mixture was partitioned between ethyl acetate and water.

Filtration through Celite was necessary to break the emulsion, after

which the collected organic was washed with water. The organic was

dried over magnesium sulfate, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation by ﬂash column chromatography (7:3

heptane/ethyl acetate) aﬀorded the title compound as a white solid

1

(7.5 g, 62%). H NMR (400 MHz, CDCl₃) δ 7.11 (s, 2H), 4.19 (m,

2H), 2.78 (m, 2H), 2.71 (m, 1H), 1.81 (m, 2H), 1.65 (m, 2H), 1.48 (s,

9H).

tert-Butyl 4-(2-Chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-

4-yl)piperidine-1-carboxylate (52). A solution of 3,3-diﬂuoropyr-

rolidine hydrochloride (533 mg, 3.72 mmol), tert-butyl 4-(2,6-

dichloropyridin-4-yl)piperidine-1-carboxylate (51) (1.00 g, 1.17

mmol), and N,N-diisopropylethylamine (1.23 mL, 7.03 mmol) in N-

methylpyrrolidinone (2.3 mL) was heated at 130 °C in the microwave

(CEM) for 1.5 h. The reaction mixture was diluted with ethyl acetate

(50 mL), and the resulting solution was washed with saturated

aqueous ammonium chloride solution (2 × 20 mL). The aqueous

washes were extracted with ethyl acetate (25 mL). The combined

organic was washed with water (25 mL), dried over anhydrous sodium

sulfate, ﬁltered, and concentrated under reduced pressure. Puriﬁcation

by ﬂash column chromatography (4:1 heptane/ethyl acetate) aﬀorded

tert-Butyl 4-(2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(4-

(triﬂuoromethyl)pyridin-2-ylamino)pyridin-4-yl)piperidine-1-

carboxylate (48). A solution of tert-butyl 4-[2-(3,3-diﬂuoropyrroli-

din-1-yl)-6-[[4-(triﬂuoromethyl)-2-pyridyl]amino]-4-pyridyl]-3,6-di-

hydro-2H-pyridine-1-carboxylate (47) (75 mg, 0.14 mmol) in

methanol (50 mL) was hydrogenated in a H-Cube (10% Pd/C

cartridge, 40 bar of hydrogen, 30 °C, ﬂow rate = 1 mL/min). LCMS

indicated incomplete reaction, and the reaction solution was further

hydrogenated (10% Pd/C cartridge, 40 bar of hydrogen, 40 °C, ﬂow

rate of 1 mL/min). The reaction solution was concentrated to aﬀord

the title compound as a light brown solid (63 mg, 84% crude yield).

LCMS: m/z = 528 [M + H]⁺.

1

the title compound as a clear oil (220 mg, 47% yield). H NMR (400

MHz, CDCl₃), δ 6.51 (s, 1 H), 6.03 (s, 1 H), 4.25 (m, 2 H), 3.83 (t, J

= 13.1 Hz, 2 H), 3.67 (t, J = 7.3 Hz, 2 H), 2.77 (m, 2 H), 2.58−2.42

(m, 3 H), 1.78 (m, 2 H), 1.62−1.53 (m, 2 H), 1.48 (s, 9 H).

2-Chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)-

piperidin-4-yl)pyridine (53). To an ice-cooled solution of tert-butyl

4-(2-chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-4-yl)piperidine-1-

carboxylate (52) (0.220 g, 0.547 mmol) in dichloromethane (2 mL)

was added triﬂuoroacetic acid (2 mL). After 2 h, the reaction mixture

was concentrated under reduced pressure. The resulting residue was

dissolved in dichloromethane (10 mL) and washed with saturated

aqueous sodium bicarbonate solution (5 mL). The organic layer was

dried over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. The resulting residue was dissolved in tetrahy-

drofuran (2 mL). Oxetan-3-one (0.112 mL, 1.10 mmol) and sodium

triacetoxyborohydride (366 mg, 1.64 mmol) were sequentially added

to the solution at 23 °C. After 35 min, the reaction mixture was

partitioned between ethyl acetate (10 mL) and saturated aqueous

ammonium chloride solution (10 mL). The organic was separated, and

the aqueous layer was further extracted with ethyl acetate (2 × 5 mL).

The combined organic was dried over anhydrous sodium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by ﬂash

column chromatography (98:2 dichloromethane/methanol + 0.1%

ammonium hydroxide) aﬀorded the title compound as a clear oil (166

6-(3,3-Diﬂuoropyrrolidin-1-yl)-4-(piperidin-4-yl)-N-(4-

(triﬂuoromethyl)pyridin-2-yl)pyridin-2-amine (49). To a solu-

tion of tert-butyl 4-[2-(3,3-diﬂuoropyrrolidin-1-yl)-6-[[4-(triﬂuoro-

methyl)-2-pyridyl]amino]-4-pyridyl]piperidine-1-carboxylate (48)

(60 mg, 0.1 mmol) in dichloromethane (1.0 mL) was slowly added

4 M HCl in 1,4-dioxane (2.0 mL) at 23 °C. After 1 h, the mixture was

concentrated under reduced pressure and dried under vacuum to

aﬀord the title compound as an oﬀ-white solid (50 mg, 86% crude

yield). LCMS: m/z = 428 [M + H]⁺.

1-(4-(2-(3,3-Diﬂuoropyrrolidin-1-yl)-6-(4-(triﬂuoromethyl)-

pyridin-2-ylamino)pyridin-4-yl)piperidin-1-yl)ethanone (20).

To a solution of 6-(3,3-diﬂuoropyrrolidin-1-yl)-4-(4-piperidyl)-N-[4-

(triﬂuoromethyl)-2-pyridyl]pyridin-2-amine (49) (0.050 g, 0.11

mmol) and N,N-diisopropylethylamine (0.051 mL, 0.29 mmol) in

tetrahydrofuran (2.0 mL) was added acetyl chloride (0.009 mL, 0.1

mmol) at 23 °C. After 1 h, the reaction mixture was diluted with ethyl

acetate (10 mL), and the resulting solution was washed with 1 N

aqueous sodium bicarbonate solution. The collected organic was dried

over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation by preparative reverse phase HPLC

1

mg, 85% yield). H NMR (CDCl₃, 400 MHz), δ: 6.54 (s, 1 H), 6.07

M

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Journal of Medicinal Chemistry

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(s, 1 H), 4.67 (t, J = 6.3 Hz, 2 H), 4.63 (t, J = 6.3 Hz, 2 H), 3.82 (t, J =

13.2 Hz, 2 H), 3.66 (t, J = 7.2 Hz, 2 H), 3.50 (m, 1 H), 2.86 (m, 2 H),

2.52−2.38 (m, 3 H), 1.94−1.72 (m, 6 H).

mol %), and sodium tert-butoxide (6 equiv) was purged under

nitrogen before the addition of anhydrous tetrahydrofuran (0.1 M).

The mixture was stirred at 90 °C for 16 h before ﬁltering through

Celite, rinsing with dichloromethane. After concentration under

reduced pressure, the reaction residue was puriﬁed by preparative

reverse phase HPLC to aﬀord the title compound.

2-((6-(3-Methoxyazetidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4-

yl)pyridin-2-yl)amino)isonicotinonitrile (28). Reaction of 3-

methoxyazetidine following the general Buchwald−Hartwig procedure

aﬀorded the title compound (47% yield). LCMS: m/z = 421 [M +

H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.77 (br s, 1H), 8.45 (s, 1H),

8.40 (d, J = 5.1 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H), 6.64 (s, 1H), 5.86

(s, 1H), 4.58−4.50 (m, 2H), 4.47−4.39 (m, 2H), 4.35 (m, 1H), 4.20−

4.09 (m, 2H), 3.80−3.66 (m, 2H), 3.44−3.35 (m, 1H), 3.26 (s, 3H),

2.82−2.72 (m, 2H), 2.41−2.32 (m, 1H), 1.88−1.76 (m, 2H), 1.76−

1.57 (m, 4H).

2-((6-(3,3-Diﬂuoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)-

piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (26, GNE-

3511). To a solution of 2-chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)-4-(1-

(oxetan-3-yl)piperidin-4-yl)pyridine (53) (216 mg, 0.603 mmol), 2-

amino-4-cyanopyridine (122 mg, 1.03 mmol), cesium carbonate (399

mg, 1.22 mmol), and 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene

(44 mg, 0.068 mmol) in 1,4-dioxane was added tris(dibenzylidene-

acetone)dipalladium(0) (28 mg, 0.031 mmol). The reaction mixture

was heated at reﬂux under nitrogen for 13 h. After cooling to room

temperature, the reaction mixture was diluted with ethyl acetate (20

mL) and washed with water (10 mL). The organic was separated, and

the aqueous wash was further extracted with ethyl acetate (2 × 10

mL). The combined organics were dried over anhydrous sodium

sulfate, ﬁltered, and concentrated under reduced pressure. Puriﬁcation

by ﬂash column chromatography (97:3 dichloromethane/methanol +

0.1% ammonium hydroxide) provided the title compound as a yellow

2-((6-(Azetidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-

2-yl)amino)isonicotinonitrile (29). Reaction of azetidine following

the general Buchwald−Hartwig procedure aﬀorded the title compound

1

(47% yield). LCMS: m/z = 391 [M + H]⁺. H NMR (400 MHz,

solid (205 mg, 77% yield). H NMR (400 MHz, DMSO-d₆), δ: 9.72

(s, 1 H), 8.46 (s, 1 H), 8.41 (d, J = 5.0 Hz, 1 H), 7.20 (dd, J = 5.1, 1.2

Hz, 1 H), 6.60 (s, 1 H), 6.00 (s, 1 H), 4.54 (t, J = 6.4 Hz, 2 H), 4.44 (t,

J = 6.0 Hz, 2 H), 3.85 (t, J = 13.2 Hz, 2 H), 3.65 (t, J = 7.2 Hz, 2 H),

3.40 (m, 1 H), 2.80 (m, 2 H), 2.56 (m, 2 H), 2.39 (m, 1 H), 1.84 (m, 2

H), 1.76−1.61 (m, 4 H). ¹³C NMR (126 MHz, DMSO-d₆) δ 157.97,

155.37, 154.60, 152.44, 149.19, 128.44 (J_CF= 245.43 Hz), 119.88,

117.38, 115.98, 113.44, 98.62, 96.98, 74.66, 58.55, 53.44 (J_CF= 31.39

Hz), 49.63, 44.08, 41.64, 38.88, 33.06 (J_CF= 23.66 Hz), 31.64. HRMS

(ESI) m/z: [M + H]⁺calcd for C₂₃H₂₇F₂N₆O, 441.2209. Found:

441.2211.

2,6-Dichloro-4-[1-(oxetan-3-yl)-4-piperidyl]pyridine (54a).

To an ice-cooled solution of tert-butyl 4-(2,6-dichloro-4-pyridyl)-

piperidine-1-carboxylate (51) (2.84 g, 8.57 mmol) in dichloromethane

(17 mL) was added triﬂuoroacetic acid (8.4 mL), and the solution was

warm to room temperature. After stirring for 1 h, the solution was

concentrated under reduced pressure to aﬀord a white solid which was

resuspended in anhydrous tetrahydrofuran (34 mL) with triethylamine

(6.0 mL, 43 mmol) and 3-oxetanone (0.82 mL, 13 mmol). After

stirring for 30 min, sodium triacetoxyborohydride (5.73 g, 25.7 mmol)

was added and stirring continued for 2 h. The reaction mixture was

diluted with dichloromethane and washed with saturated aqueous

sodium bicarbonate. The collected organic was dried over magnesium

sulfate, ﬁltered, and concentrated under reduced pressure to aﬀord title

compound as a beige solid (2.67 g, >99% crude yield over 2 steps). ¹H

NMR (400 MHz, CDCl₃) δ 7.13 (s, 2H), 4.73−4.63 (m, 4H), 3.60−

3.52 (m, 1H), 3.00−2.88 (m, 2H), 2.60−2.48 (m, 1H), 2.05−1.94 (m,

2H), 1.93−1.78 (m, 4H).

DMSO-d₆) δ 9.76 (br s, 1H), 8.51 (s, 1H), 8.40 (d, J = 5.1 Hz, 1H),

7.18 (d, J = 5.1 Hz, 1H), 6.59 (s, 1H), 5.80 (s, 1H), 4.57−4.49 (m,

2H), 4.49−4.40 (m, 2H), 4.00−3.92 (m, 4H), 3.43−3.35 (m, 1H),

2.82−2.73 (m, 2H), 2.40−2.27 (m, 3H), 1.88−1.78 (m, 2H), 1.77−

1.68 (m, 2H), 1.68−1.51 (m, 2H).

2-((6-Cyclobutoxy-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-

2-yl)amino)isonicotinonitrile (30). A vial charged with 6-chloro-N-

(4-(diﬂuoromethyl)pyridin-2-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)-

pyridin-2-amine (55) (0.040 g, 0.11 mmol), palladium(II) acetate (2.4

mg, 0.011 mmol), 5-di(1-adamantylphosphino)-1-(1,3,5-triphenyl-1H-

pyrazol-4-yl)-1H-pyrazole (13 mg, 0.022 mmol), and cesium carbonate

(106 mg, 0.325 mmol) was purged with nitrogen before the addition

of 3-cyclobutanol (86 μL, 1.1 mmol) and anhydrous toluene (1.1 mL).

The mixture was stirred at 120 °C for 16 h. The mixture was diluted

with dichloromethane, ﬁltered through Celite (rinsing with dichloro-

methane). The organic was dried over magnesium sulfate, ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed by

preparative reverse phase HPLC to aﬀord the title compound as a

colorless solid (28 mg, 64% yield). LCMS: m/z = 406.3 [M + H]⁺. ¹H

NMR (400 MHz, DMSO-d₆) δ 9.98 (br s, 1H), 8.50−8.39 (m, 2H),

7.25 (dd, J = 5.1, 1.3 Hz, 1H), 6.83 (s, 1H), 6.20 (s, 1H), 5.11 (m,

1H), 4.57−4.50 (m, 2H), 4.47−4.41 (m, 2H), 3.40 (m, 1H), 2.79 (d, J

= 11.3 Hz, 2H), 2.47−2.37 (m, 3H), 2.18−2.02 (m, 2H), 1.88−1.79

(m, 3H), 1.78−1.70 (m, 2H), 1.70−1.54 (m, 3H).

General Procedure for Suzuki−Miyaura Reaction Triﬂuor-

oborate Potassium Salts. A vial charged with the 2-((6-chloro-4-(1-

(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55)

(1 equiv), palladium(II) acetate (10 mol %), butyldi-1-adamantyl-

phosphine (15 mol %), the potassium triﬂuoroborate salt (1.2 equiv),

and cesium carbonate (3 equiv) was purged under nitrogen before the

addition of degassed toluene (0.2 M) and degassed water (2 M). The

mixture was stirred at 110 °C overnight and then diluted with

dichloromethane, ﬁltered through Celite (rinsing with dichloro-

methane). The organics were dried over magnesium sulfate, ﬁltered,

and concentrated under reduced pressure. The reaction residue thus

obtained was puriﬁed by preparative reverse phase HPLC to aﬀord the

title compound.

2-((6-Chloro-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)-

amino)isonicotinonitrile (55a). To 2,6-dichloro-4-[1-(oxetan-3-yl)-

4-piperidyl]pyridine (0.720 g, 2.51 mmol), 2-amino-5-cyanopyridine

(308 mg, 2.51 mmol), tris(dibenzylideneacetone)dipalladium(0) (59

mg, 0.064 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

(0.090 g, 0.16 mmol), and cesium carbonate (1.14 g, 3.51 mmol) was

added anhydrous 1,4-dioxane (10 mL) under nitrogen. The reaction

mixture was stirred at 80 °C for 20 h before cooling to room

temperature and ﬁltering through Celite (rinsing with dichloro-

methane). After concentration under reduced pressure, ﬂash column

chromatography (9:1 dichloromethane/methanol) aﬀorded the title

compound as a colorless solid (725 mg, 78% yield). LCMS: m/z = 370

2-((6-Ethyl-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)-

amino)isonicotinonitrile (32). Reaction of ethyl triﬂuoroborate

potassium salt with 6-chloro-N-(4-(diﬂuoromethyl)pyridin-2-yl)-4-(1-

(oxetan-3-yl)piperidin-4-yl)pyridin-2-amine (55) (50.0 mg, 0.135

mmol) following the general Suzuki−Miyaura procedure aﬀorded

the title compound as a colorless solid (25 mg, 50% yield). LCMS: m/

z = 364 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.99 (br s, 1H),

8.44 (d, J = 5.0 Hz, 1H), 8.35 (s, 1H), 7.27 (s, 1H), 7.21 (dd, J = 5.1,

1.4 Hz, 1H), 6.75 (s, 1H), 4.57−4.50 (m, 2H), 4.46−4.42 (m, 2H),

3.47−3.36 (m, 1H), 2.86−2.74 (m, 2H), 2.69 (q, J = 7.5 Hz, 2H),

2.48−2.41 (m, 1H), 1.91−1.81 (m, 2H), 1.81−1.72 (m, 2H), 1.72−

1.56 (m, 2H), 1.26 (t, J = 7.6 Hz, 3H).

1

[M + H]⁺. H NMR (400 MHz, DMSO-d₆) δ 10.38 (br s, 1H), 8.49

(d, J = 5.1 Hz, 1H), 7.94 (s, 1H), 7.63 (s, 1H), 7.30 (d, J = 5.1 Hz,

1H), 6.98 (s, 1H), 4.57−4.51 (m, 2H), 4.47−4.41 (m, 2H), 3.46−3.38

(m, 1H), 2.85−2.77 (m, 2H), 1.90−1.75 (m, 4H), 1.70−1.58 (m, 2H).

General Procedure for Buchwald−Hartwig Reaction of

Secondary Amines. A vial charged with the 2-((6-chloro-4-(1-

(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55)

(1 equiv), the amine (usually as the HCl salt, 3 equiv), chloro(2-

dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2-

aminoethylphenyl)]palladium(II) methyl-tert-butyl ether adduct (10

mol %), 2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl (10

N

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2-((6-Cyclopropyl-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-

yl)amino)isonicotinonitrile (33). Reaction of cyclopropyl triﬂuor-

oborate potassium salt with 6-chloro-N-(4-(diﬂuoromethyl)pyridin-2-

yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-amine (55) (50.0 mg,

0.135 mmol) following the general Suzuki−Miyaura procedure

aﬀorded the title compound as a colorless solid (8.5 mg, 17% yield).

LCMS: m/z = 376 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.91

(s, 1H), 8.42 (d, J = 5.0 Hz, 1H), 8.34 (s, 1H), 7.22 (dd, J = 5.1, 1.3

Hz, 1H), 7.07 (s, 1H), 6.82 (d, J = 0.9 Hz, 1H), 4.58−4.52 (m, 2H),

4.47−4.42 (m, 2H), 3.46−3.36 (m, 1H), 2.84−2.75 (m, 2H), 2.47−

2.38 (m, 1H), 2.07−1.98 (m, 1H), 1.90−1.80 (m, 2H), 1.80−1.72 (m,

2H), 1.72−1.57 (m, 2H), 0.98−0.90 (m, 4H).

(57) (100 mg, 0.33 mmol) following the procedure for the preparation

of tert-butyl 4-(2-(3,3-diﬂuoropyrrolidin-1-yl)-6-(4-(triﬂuoromethyl)-

pyridin-2-ylamino)pyridin-4-yl)-5,6-dihydropyridine-1(2H)-carboxy-

late (47) aﬀorded the title compound as an oﬀ-white solid (37 mg,

1

29% yield). LCMS: m/z = 386 [M + H]⁺. H NMR (400 MHz,

DMSO-d₆) δ 9.84 (s, 1H), 8.47 (s, 1H), 8.41 (d, J = 5.0 Hz, 1H), 7.21

(d, J = 5.0 Hz, 1H), 6.61 (s, 1H), 6.00 (s, 1H), 3.94 (d, J = 10.9 Hz,

2H), 3.86 (t, J = 13.2 Hz, 2H), 3.65 (t, J = 7.2 Hz, 2H), 3.47−3.38 (m,

2H), 2.70−2.52 (m, 3H), 1.71−1.62 (m, 4H).

tert-Butyl 3-(2,6-Dichloropyridin-4-yl)azetidine-1-carboxy-

late (58). Reaction of 1-Boc-3-(iodo)azetidine (11.3 g, 39.0 mmol)

following the procedure for the preparation of tert-butyl 4-(2,6-

dichloro-4-pyridyl)piperidine-1-carboxylate (51) aﬀorded the title

2-((6-Methyl-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)-

amino)isonicotinonitrile (31). 31 was made from tert-butyl 4-(2-

chloro-6-methyl-4-pyridyl)piperidine-1-carboxylate via Buchwald−

Hartwig, Boc deprotection, and reductive amination as previously

1

compound (1.73 g, 54% yield). LCMS: m/z = 303 [M + H]⁺. H

NMR (400 MHz, CDCl₃) δ: 7.23 (s, 2H), 4.35 (t, J = 8.7 Hz, 2H),

3.92 (dd, J = 8.7, 5.6 Hz, 2H), 3.73−3.61 (m, 1H), 1.47 (s, 9H). ¹³C

NMR (101 MHz, CDCl₃) δ 157.03, 156.08, 151.09, 121.24, 80.24,

55.31, 32.48, 28.32.

2,6-Dichloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridine (59).

Reaction of tert-butyl 3-(2,6-dichloropyridin-4-yl)azetidine-1-carbox-

ylate (58) (940 mg, 3.10 mmol) following the procedure for the

preparation of 2-chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)-4-(1-(oxetan-3-

yl)piperidin-4-yl)pyridine (53) except that the reductive amination

was performed at 50 °C aﬀorded the title compound as a red oil (640

mg, 80% over two steps). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (s, 2H),

4.75−4.68 (m, 2H), 4.57−4.52 (m, 2H), 3.82−3.77 (m, 1H), 3.77−

3.71 (m, 2H), 3.67−3.58 (m, 1H), 3.32−3.27 (m, 2H).

2-((6-Chloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridin-2-yl)-

amino)isonicotinonitrile (60). Reaction of 2,6-dichloro-4-(1-(ox-

etan-3-yl)azetidin-3-yl)pyridine (58) (1.50 g, 5.79 mmol) following

the procedure for the preparation of 2-((6-chloro-4-(1-(oxetan-3-

yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile (55) aﬀorded

the title compound as a white solid (1.0 g, 51%). ¹H NMR (400 MHz,

CDCl₃) δ 8.39 (dd, J = 5.0, 0.8 Hz, 1H), 7.97 (d, J = 0.8 Hz, 1H), 7.54

(br s, 1H), 7.29 (s, 1H), 7.09 (dd, J = 5.1, 1.3 Hz, 1H), 6.96 (s, 1H),

4.76−4.69 (m, 2H), 4.61−4.53 (m, 2H), 3.85−3.79 (m, 1H), 3.79−

3.72 (m, 2H), 3.71−3.60 (m, 1H), 3.34−3.29 (m, 2H).

reported (WO 2013174780).²²LCMS: m/z = 350 [M + H]⁺. H

1

NMR (400 MHz, DMSO-d₆) δ 9.99 (br s, 1H), 8.44 (d, J = 5.1 Hz,

1H), 8.18 (s, 1H), 7.36 (s, 1H), 7.21 (d, J = 5.1 Hz, 1H), 6.75 (s, 1H),

4.54 (dd, J = 6.5, 6.5 Hz, 2H), 4.45 (dd, J = 6.5, 6.5 Hz, 2H), 3.42 (m,

1H), 2.80 (m, 2H), 2.44 (m, 1H), 2.40 (s, 3H), 1.86 (m, 2H), 1.76 (m,

2H), 1.63 (m, 2H).

2-((4-(1-Acetylpiperidin-4-yl)-6-chloropyridin-2-yl)amino)-

isonicotinonitrile (55b). A solution of tert-butyl 4-(2,6-dichloro-4-

pyridyl)piperidine-1-carboxylate (51) (1.79 g, 5.41 mmol) in triﬂuoro-

acetic acid (5.4 mL) was stirred for 1 h before the solution was

concentrated under reduced pressure to aﬀord the triﬂuoroacetate salt

as a white solid. Dissolution of the residue in anhydrous dichloro-

methane, (11 mL, 0.5 M) was followed by the addition of

triethylamine (2.3 mL, 16 mmol), 4-dimethylaminopyridine (33 mg,

0.27 mmol), and acetic anhydride (0.79 mL, 8.1 mmol). After stirring

3.5 h, the reaction mixture was diluted with dichloromethane and

washed with saturated aqueous sodium bicarbonate solution. The

collected organic was dried over magnesium sulfate, ﬁltered, and

concentrated under reduced pressure. Three quarters of the crude

material was subjected to same procedure as the synthesis of 2-((6-

chloro-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)-

isonicotinonitrile (55a) employing 2-amino-5-cyanopyridine to aﬀord

the title compound as a white solid (1.07 g, 74% over three steps).

LCMS: m/z = 356 [M + H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.40 (d,

J = 5.0 Hz, 1H), 7.90 (s, 1H), 7.39 (br s, 1H), 7.20 (s, 1H), 7.08 (d, J

= 5.0 Hz, 1H), 6.81 (s, 1H), 4.87−4.78 (m, 1H), 4.00−3.92 (m, 1H),

3.22−3.13 (m, 1H), 2.79−2.70 (m, 1H), 2.68−2.57 (m, 1H), 2.14 (s,

3H), 1.99−1.86 (m, 2H), 1.69−1.58 (m, 2H).

2-((6-(3,3-Diﬂuoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)azetidin-

3-yl)pyridin-2-yl)amino)isonicotinonitrile (27). Reaction of 2-((6-

chloro-4-(1-(oxetan-3-yl)azetidin-3-yl)pyridin-2-yl)amino)-

isonicotinonitrile (50.0 mg, 0.146 mmol) following the general

Buchwald−Hartwig procedure aﬀorded the title compound as a

1

white solid (15 mg, 25%). LCMS: m/z = 413 [M + H]⁺. H NMR

(400 MHz, DMSO-d₆) δ 9.83 (br s, 1H), 8.47 (s, 1H), 8.41 (d, J = 5.0

Hz, 1H), 7.21 (d, J = 5.0 Hz, 1H), 6.71 (s, 1H), 6.02 (s, 1H), 4.62−

4.53 (m, 2H), 4.44−4.36 (m, 2H), 3.93−3.80 (m, 2H), 3.80−3.71 (m,

1H), 3.70−3.59 (m, 4H), 3.59−3.48 (m, 1H), 3.24−3.18 (m, 2H),

2.63−2.53 (m, 2H).

2-(4-(1-Acetylpiperidin-4-yl)-6-(3,3-diﬂuoropyrrolidin-1-yl)-

pyridin-2-ylamino)isonicotinonitrile (21). Reaction of 3,3-diﬂuor-

opyrrolidine hydrochloride and 2-((4-(1-acetylpiperidin-4-yl)-6-chlor-

opyridin-2-yl)amino)isonicotinonitrile (55b) following the general

1

Buchwald−Hartwig procedure aﬀorded the title compound. H NMR

4-(2,6-Dichloro-4-pyridyl)tetrahydropyran-4-carbonitrile

(62). To a stirring solution of 2,4,6-trichloropyridine (4.65 g, 24.7

mmol) and tetrahydropyran-4-carbonitrile (2.29 g, 20.6 mmol) in

tetrahydrofuran (100 mL, 0.2 M) at −78 °C under nitrogen was added

lithium bis(trimethylsilyl) amide (29 mL, 29 mmol, 1.0 M in

tetrahydrofuran). After 5 min, the cooling bath was removed. After

stirring further for 40 min, the reaction was quenched by the addition

of saturated aqueous ammonium chloride solution. The organic was

removed under reduced pressure. The resulting mixture was extracted

with dichloromethane. The collected organic was dried over

magnesium sulfate, ﬁltered, and concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (7:3 heptane/ethyl

acetate) aﬀorded the title compound as a white solid (3.75 g, 71%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.39 (d, J = 0.6 Hz, 2H), 4.17−

4.05 (m, 2H), 3.93−3.84 (m, 2H), 2.15−1.99 (m, 4H).

2-((6-Chloro-4-(4-cyanotetrahydro-2H-pyran-4-yl)pyridin-2-

yl)amino)isonicotinonitrile (64). To 4-(2,6-dichloro-4-pyridyl)-

tetrahydropyran-4-carbonitrile (62) (1.50 g, 5.83 mmol), 2-amino-5-

cyanopyridine (716 mg, 5.83 mmol), tris(dibenzylideneacetone)-

dipalladium(0) (138 mg, 0.151 mmol), 2,2′-bis(diphenylphosphino)-

1,1′-binaphthyl (187 mg, 0.300 mmol), and cesium carbonate (2.66 g,

8.17 mmol) was added anhydrous 1,4-dioxane (23 mL, 0.25 M) under

(400 MHz, DMSO-d₆) δ 9.81 (s, 1H), 8.51−8.43 (m, 1H), 8.41 (d, J =

5.0 Hz, 1H), 7.20 (dd, J = 5.0, 1.4 Hz, 1H), 6.58 (s, 1H), 6.00 (s, 1H),

4.59−4.45 (m, 1H), 4.01−3.77 (m, 3H), 3.71−3.55 (m, 2H), 3.19−

3.01 (m, 1H), 2.71−2.54 (m, 4H), 2.03 (s, 3H), 1.85−1.68 (m, 2H),

1.67−1.34 (m, 2H).

2,6-Dichloro-4-(tetrahydro-2H-pyran-4-yl)pyridine (56). Re-

action of 4-bromotetrahydro-2H-pyran (2.88 mL, 25.6 mmol)

following the procedure for the preparation of tert-butyl 4-(2,6-

dichloro-4-pyridyl)piperidine-1-carboxylate (51) aﬀorded the title

1

compound as a white solid (3.0 g, 70% yield). H NMR (CDCl₃,

400 MHz) δ 7.11 (s, 1H), 4.09 (m, 2H), 3.57−3.42 (m, 2H), 2.85−

2.70 (m, 1H), 1.77 (m, 4H).

2-Chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)-4-(tetrahydro-2H-

pyran-4-yl)pyridine (57). Reaction of 2,6-dichloro-4-(tetrahydro-

2H-pyran-4-yl)pyridine (1.20 g, 5.20 mmol) following the procedure

for the preparation of tert-butyl 4-(2-chloro-6-(3,3-diﬂuoropyrrolidin-

1-yl)pyridin-4-yl)piperidine-1-carboxylate (52) aﬀorded the title

compound. LCMS: m/z = 303 [M + H]⁺.

2-(6-(3,3-Diﬂuoropyrrolidin-1-yl)-4-(tetrahydro-2H-pyran-4-

yl)pyridin-2-ylamino)isonicotinonitrile (22). Reaction of 2-chloro-

6-(3,3-diﬂuoropyrrolidin-1-yl)-4-(tetrahydro-2H-pyran-4-yl)pyridine

O

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

nitrogen. The reaction mixture was stirred at 80 °C for 18 h before

cooling to room temperatue and ﬁltering through Celite. After

concentration under reduced pressure, puriﬁcation by ﬂash column

chromatography (1:1 heptane/ethyl acetate) aﬀorded the title

compound as a colorless solid (880 mg, 44% yield). LCMS: m/z =

(1.0 g, 7.3 mmol), and N,N-diisopropylethylamine (1.88 g, 14.6

mmol) in dimethyl sulfoxide (10 mL) was heated between 80 and 90

°C for 16 h. After cooling to room temperature, the mixture was

poured into water (10 mL), and the resulting mixture was extracted

with ethyl acetate (3 × 20 mL). The combined organic was

concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography aﬀorded the title compound as a white solid (0.20 g,

50% yield). LCMS: m/z = 276.9 [M + H]⁺,

2-(6-(3,3-Diﬂuoropyrrolidin-1-yl)-4-(2-hydroxypropan-2-yl)-

pyridin-2-ylamino)isonicotinonitrile (25). To a mixture of 2-(2-

chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-4-yl)propan-2-ol (68)

(0.10 g, 0.36 mmol), 2-amino-4-cyanopyridine (86.3 mg, 0.725

mmol), and K₃PO₄(155 mg, 0.72 mmol) in anhydrous 1,4-dioxane

(15 mL) was added Pd(t-Bu₃P)₂(18 mg, 0.036 mmol) under nitrogen.

The resulting mixture was heated to reﬂux for 3 h. After cooling to

room temperature, the mixture was ﬁltered and concentrated under

reduced pressure. Puriﬁcation by preparative reverse phase HPLC

aﬀorded the title compound as a white solid (0.080 g, 62% yield).

LCMS: m/z = 359.9 [M + H]⁺. ¹H NMR (400 MHz, CD₃OD) δ 8.52

(s, 1H), 8.34−8.33 (m, 1H), 7.06−7.04 (m, 1H), 6.63 (s, 1H), 6.19 (s,

1H), 3.99 (t, J = 13.2 Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 2.60−2.49 (m,

2H), 1.51 (s, 6H).

1

340 [M + H]⁺. H NMR (400 MHz, CDCl₃) δ 8.44 (d, J = 5.1 Hz,

1H), 7.82 (s, 1H), 7.67 (s, 1H), 7.52 (br s, 1H), 7.13 (d, J = 5.1 Hz,

1H), 7.05 (s, 1H), 4.21−4.07 (m, 2H), 3.97−3.79 (m, 2H), 2.26−2.08

(m, 2H), 2.08−1.99 (m, 2H).

2-(4-(4-Cyanotetrahydro-2H-pyran-4-yl)-6-(3,3-diﬂuoropyr-

rolidin-1-yl)pyridin-2-ylamino)isonicotinonitrile (23). Reaction

of 3,3-diﬂuoropyrrolidine hydrochloride with 2-((6-chloro-4-(4-

cyanotetrahydro-2H-pyran-4-yl)pyridin-2-yl)amino)isonicotinonitrile

(64) following the general Buchwald−Hartwig procedure aﬀorded the

title compound (47% yield). LCMS: m/z = 411.2 [M + H]⁺. ¹H NMR

(400 MHz, DMSO-d₆) δ 10.01 (br s, 1H), 8.47−8.41 (m, 2H), 7.29−

7.22 (m, 1H), 6.93 (d, J = 0.9 Hz, 1H), 6.18 (d, J = 1.0 Hz, 1H), 4.08−

3.97 (m, 2H), 3.97−3.84 (m, 2H), 3.76−3.60 (m, 4H), 2.67−2.56 (m,

2H), 2.17−1.98 (m, 4H).

2-(2,6-Dichloropyridin-4-yl)-2-methylpropanenitrile (63). To

a solution of 2,4,6-trichloropyridine (6.0 g, 33 mmol) and 2-

methylpropanenitrile (1.9 g, 28 mmol) in anhydrous tetrahydrofuran

(30 mL) was added dropwise lithium bis(trimethylsilyl)amide (38.6

mL, 38.6 mmol, 1 M in tetrahydrofuran) at −78 °C under nitrogen.

After 15 min, the reaction mixture was warmed to room temperature

for 2 h. The reaction mixture was diluted with saturated aqueous

ammonia chloride solution (30 mL), and the resulting solution was

extracted with ethyl acetate (3 × 60 mL). The combined organic was

concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography aﬀorded the title compound as a white solid (6.0 g,

84% yield). LCMS: m/z = 214.7 [M + H]⁺.

2-(2-Chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-4-yl)-2-

methylpropanenitrile (65). A mixture of 2-(2,6-dichloropyridin-4-

yl)-2-methylpropanenitrile (63) (0.30 g, 1.4 mmol), 3,3-diﬂuoropyr-

rolidine hydrochloride (1 g, 7 mmol), and N,N-diisopropylethylamine

(1.8 g, 14 mmol) in dimethyl sulfoxide (20 mL) was heated between

80−90 °C for 16 h. After cooling to room temperature, the reaction

solution was poured into water (10 mL), and the resulting mixture was

extracted with ethyl acetate (3 × 20 mL). The collected organic was

concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography aﬀorded the title compound as a white solid (0.20 g,

50% yield). LCMS: m/z = 285.8 [M + H]⁺.

AUTHOR INFORMATION

Corresponding Authors

■

*J.W.L.: phone, 650-467-2877; e-mail, lewcock.joseph@gene.

com.

*M.S.: phone, 650-467-7764; e-mail, siu.michael@gene.com.

Present Addresses

^#F.C.: Achaogen, 7000 Shoreline Court, No. 371, South San

Francisco, CA 94080, U.S.

^∞C.E.L.P.: National Institute for Neurological Disorders and

Stroke, National Institutes of Health, 35A Convent Drive,

Bethesda, MD 20892, U.S.

^×J.P.L.: Biogen Idec, 14 Cambridge Center, Cambridge, MA

02142, U.S.

^○Y.G.S.: College of Pharmacy, Chungnam National University,

99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of

Korea (South).

^△B.W.: Well Institute for Cell and Molecular Biology, Cornell

University, Ithaca, NY, U.S.

2-(4-(2-Cyanopropan-2-yl)-6-(3,3-diﬂuoropyrrolidin-1-yl)-

pyridin-2-ylamino)isonicotinonitrile (24). To a solution of 2-(2-

chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-4-yl)-2-methylpropaneni-

trile (65) (0.20 g, 0.70 mmol), 2-amino-4-cyanopyridine (167 mg, 1.40

mmol), and K₃PO₄(0.30 g, 1.4 mmol) in anhydrous 1,4-dioxane (15

mL) was added Pd(t-Bu₃P)₂(36 mg, 0.070 mmol) under nitrogen.

The resulting solution was heated to reﬂux for 3 h. After cooling to

room temperature, the mixture was ﬁltered and concentrated under

reduced pressure. Puriﬁcation by preparative reverse phase HPLC

aﬀorded the title compound as a white solid (11 mg, 4.2% yield).

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the following individuals for their contributions:

Chris Hamman, Mengling Wong, and Michael Hayes for

compound puriﬁcation; Emile Plise and Jonathan Cheong for

MDR1-MDCK data; Xiaolin Zhang, Allan Jaochico, and Xiao

Ding for bioanalytical data; Amy Sambrone for formulations

work; York Rudhard and the Evotec team for biochemical and

cell-based potency data; Wuxi for support with analog

synthesis; and Genentech compound management for sample

handling.

1

LCMS: m/z 368.9 [M + H]⁺. H NMR (400 MHz, CDCl₃) δ 8.38−

8.35 (m, 2H), 7.40 (s, 1H), 7.03 (d, J = 5.2 Hz, 1H), 6.44 (s, 1H), 6.03

(s, 1H), 3.87 (t, J = 13.2 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 2.59−2.48

(m, 2H), 1.71 (s, 6H).

2-(2,6-Dichloropyridin-4-yl)propan-2-ol (67). To a solution of

methyl 2,6-dichloroisonicotinate (61) (1.0 g, 4.9 mmol) in

tetrahydrofuran (30 mL) was added CH₃MgBr (4.1 mL, 12.3 mmol,

3 M in tetrahydrofuran) dropwise at −78 °C under nitrogen. After 1 h,

the mixture was warmed to room temperature for 1 h. The reaction

mixture was diluted with saturated aqueous ammonia chloride solution

(15 mL), and the resulting mixture was extracted with ethyl acetate (3

× 30 mL). The collected organic was concentrated under reduced

pressure. Puriﬁcation by ﬂash column chromatography aﬀorded the

title compound as a white solid (700 mg, 70% yield). LCMS: m/z =

205.7 [M + H]⁺.

ABBREVIATIONS USED

■

CDI, carbonyldiimidazole; ClogD, calculated logarithm of

distribution coeﬃcient; CNS MPO, central nervous system

multiparameter optimization; cpK_a, calculated pK_a; DMEM,

Dulbecco’s modiﬁed Eagle medium; DLK, dual leucine zipper

kinase; DRG, dorsal root ganglion; FBS, fetal bovine serum; IP,

intraperitoneal; JNK, c-Jun N-terminal kinase; LipE, lipophilic

ligand eﬃciency; MAP3K12, mitogen-activated protein kinase

kinase kinase 12; MCT, methylcellulose Tween 80; MKK,

2-(2-Chloro-6-(3,3-diﬂuoropyrrolidin-1-yl)pyridin-4-yl)-

propan-2-ol (68). A solution of 2-(2,6-dichloropyridin-4-yl)propan-

2-ol (67) (0.300 g, 1.46 mmol), 3,3-diﬂuoropyrrolidine hydrochloride

P

dx.doi.org/10.1021/jm5013984 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors with activity in neurodegeneration models

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