Scaffold-Hopping and Structure-Based Discovery of Potent, Selective, And Brain Penetrant N-(1H-Pyrazol-3-yl)pyridin-2-amine Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12)

Article

pubs.acs.org/jmc

Scaﬀold-Hopping and Structure-Based Discovery of Potent,

Selective, And Brain Penetrant N‑(1H‑Pyrazol-3-yl)pyridin-2-amine

Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12)

Snahel Patel,*^,†Seth F. Harris,*^,§Paul Gibbons,^†Gauri Deshmukh,^⊥Amy Gustafson,^∇Terry Kellar,^†

Han Lin,^‡Xingrong Liu,^⊥Yanzhou Liu,^†Yichin Liu,^∇Changyou Ma,^#Kimberly Scearce-Levie,^‡

Arundhati Sengupta Ghosh,^‡Young G. Shin,^⊥,○Hilda Solanoy,^‡Jian Wang,^#Bei Wang,^‡,◆Jianping Yin,^§

Michael Siu,^†and Joseph W. Lewcock^‡

^†Departments of Discovery Chemistry, ^§Structural Biology, ^‡Neurosciences, ^⊥Drug Metabolism and Pharmacokinetics, ^∇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

S

* Supporting Information

ABSTRACT: Recent data suggest that inhibition of dual

leucine zipper kinase (DLK, MAP3K12) has therapeutic

potential for treatment of a number of indications ranging

from acute neuronal injury to chronic neurodegenerative

disease. Thus, high demand exists for selective small molecule

DLK inhibitors with favorable drug-like properties and good

CNS penetration. Herein we describe a shape-based scaﬀold

hopping approach to convert pyrimidine 1 to a pyrazole core

with improved physicochemical properties. We also present

the ﬁrst crystal structures of DLK. By utilizing a combination of property and structure-based design, we identiﬁed inhibitor 11, a

potent, selective, and brain-penetrant inhibitor of DLK with activity in an in vivo nerve injury model.

6). To address this, a parallel scaﬀold hopping^14,15strategy was

adopted (Figure 1, Approach 2) in order to improve the

There has been longstanding interest in the development of c-

properties of these compounds for development toward a

Jun N-terminal kinase (JNK) pathway inhibitors for the

potential therapeutic.¹⁶

INTRODUCTION

■

treatment of neurodegenerative disease due to the potent

neuroprotection observed in multiple contexts following

attenuation of JNK signaling.^1,2Dual leucine zipper kinase

(DLK, MAP3K12) is an upstream regulator of JNK signaling

whose expression is enriched in neurons.^3,4Recent work has

demonstrated that DLK regulates stress-induced JNK signaling

following neuronal injury and therefore represents an attractive

approach for modifying JNK activity in the central nervous

system (CNS).^5,6Consistent with this idea, genetic deletion or

pharmacological inhibition of DLK is suﬃcient to provide

considerable protection of neurons from degeneration in a

variety of in vitro and in vivo models, suggesting this kinase

represents an attractive therapeutic target.⁵⁻¹²

RESULTS AND DISCUSSION

■

To gain a speciﬁc and detailed understanding of the binding

mode of our inhibitors, we determined the novel crystal

structure of the DLK kinase domain with and without bound

ligands. The protein shows the expected canonical kinase fold,

with the ATP binding site interposed between a smaller N-

terminal subdomain and a larger, predominantly α-helical C-

terminal lobe (Figure 2). In the common model of kinase

activation, phosphorylation events trigger concerted motions of

the activation loop, helix C, and the conserved DFG motif.¹⁷⁻¹⁹

Active kinase conformations are characterized by a DFG “in”

orientation with concomitant formation of a salt bridge

between an N-terminal lysine (DLK Lys152) and its partner

acidic residue on helix C. In our apo DLK structure, the

activation loop is ordered including an α-helical segment at its

C-terminal region (residues 264−274). The DFG motif

(residues 254−256) has an active-like “in” conformation;

We recently disclosed the ﬁrst potent, selective, and brain-

penetrant compounds speciﬁcally designed to inhibit DLK. The

lead inhibitor 2 (GNE-3511, Figure 1, Approach 1), displayed

protection of primary neurons in an in vitro axon degeneration

assay as well as activity in the mouse models of glaucoma/optic

neuropathy (optic nerve crush) and Parkinson’s disease

(MPTP) after oral dosing.¹³Despite the attributes of 1 and

2, these compounds suﬀered from poor physicochemical

properties leading to high free (unbound) clearances (Table

Received: July 9, 2015

A

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Figure 1. Pyrimidine core 1 a key juncture where Approaches 1 and 2 were developed in parallel. Approach 1 resulting in pyridyl core 2 as previously

reported. Approach 2 shows the intramolecular aminopyridine C3−H interaction with pyrazole core N2 giving an example of a planar system

retained when compared to compound 1.

notable, though it remains unclear what speciﬁc role this highly

unusual variation would have in the physiological environment.

The ATP binding pocket is characterized by the classical

kinase hinge backbone pattern of hydrogen-bond interactions

on one side, while the malleable glycine-rich, “P-loop” motif

(residues 129−142) shapes both the upper surface and

opposite wall of the cavity. The back region is aﬀected by

side chain positions of the gatekeeper Met190 and the catalytic

Asp254. The binding cleft widens to the “front” toward greater

solvent exposure in the context of our truncated kinase domain

construct. This novel kinase structure serves to elucidate key

relationships of the conserved architecture and as an important

tool to guide structure-based drug design. The interactions of

our inhibitors with these features are further described below,

Figure 2. Apo crystal structure of the DLK kinase domain shows an

ordered activation segment, a DFG-in type conformation, and an

speciﬁcally contact to DLK’s adaptable P-loop as we designed

these alternate scaﬀolds.

unusual disrupted helix C. (A) The canonical kinase N- and C-

Our starting point, as exempliﬁed by 1 (DLK K_i= 0.033 μM,

terminal lobes are labeled with conserved features colored:

p-JNK IC₅₀= 0.300 μM, clogP 4.6, tPSA 74 Å², HBD 1, LipE

interdomain hinge (navy), glycine-rich P-loop (brown), helix C

(cyan), conserved N-lobe Lys152 (cyan), gatekeeper Met190 (yellow),

2.9) has low lipophilic ligand eﬃciency^16,20and poor

physiochemical properties that led to high rat-free clearance

(CL_u) and low free drug exposure (Table 6). In an attempt to

improve this, we took an alternative approach by investigating a

shape-based scaﬀold hopping strategy²¹with the goal of

replacing the central pyrimidine core with ﬁve-membered

heterocycles.²²From our assessment, the pyrazole, thiazole, and

imidazole isomers shown in Figure 1 can maintain the required

coplanar subunit similar to N-(pyridin-2-yl)pyrimidin-4-amine

1 (see Figure S2 of the Supporting Information). Further

elaboration of these analogs by the inclusion of functional

groups to extend toward the P-loop and solvent led us to the

compounds highlighted in Table 1. While each system had

comparable TanimotoShape and TanimotoCombo scores,^23,24

we initiated chemistry eﬀorts with the pyrazole core based on

synthetic tractability to expand SAR under the P-loop and

solvent regions. Attention to properties necessary for brain

penetration (HBD < 2, tPSA < 90 Å², CNS MPO > 4),^25,26

while tracking improvements to lipophilic eﬃciency, was

important to our chemistry eﬀorts. Additionally, selectivity

against the JNK pathway kinases (DLK→ MKK4/7 → JNK2/3

DFG motif (pink), activation loop (orange). (B) An active site view

shows the DFG motif (pink) “in” conformation, while the active-

kinase N-lobe Lys152 to helix C Asp161 salt bridge is not formed.

DLK’s unusual helix C Asp161 substitution (instead of conserved Glu)

is conspicuously turned out from the body of the kinase and disrupts

the helical secondary structure, creating a 13.5 Å gap between the

putative activation partners (see Supporting Information Figure S1 for

electron density of this region).

however the helix C salt bridge is not formed. DLK is unusual

in that the highly conserved acidic Glu of helix C is replaced by

an Asp substitution. This Asp161 is part of an unexpected kink

of the secondary structure that disrupts the α-helix orienting

the acidic side chain of this residue away from the body of the

kinase, ∼13 Å from its putative partner Lys152 (see Figure S1

of the Supporting Information). A lack of any crystal symmetry

mates in the vicinity suggests the conformation is not

artifactual. Given the important role of helix C in kinase

activation and the consistent disposition across all of our DLK

structures, this disrupted and inverted Asp conformation is

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a

269), while the outward, helix-disrupting orientation of helix C

Asp161 is maintained. The triﬂuoromethyl moiety makes van

der Waals contacts with the Met190 gatekeeper residue and

with Val139. To accommodate the triﬂuoromethyl and

isopropyl groups, the P-loop is lifted slightly relative to the

apo structure and bent more sharply at residues Ser133 and

Ala138 such that the tip drapes over the outer edge of the

ligand to form an enclosed cavity. The P-loop tip Gln136 is

stabilized adjacent to the ligand triﬂuoromethyl group via a

hydrogen bond to Ser253 and packs between the conserved

kinase residues Lys152 and the catalytic Asp254. Further

evaluation under the P-loop led us to introduce the proposed

cyclopentyl group from our ROCS analysis at pyrazole N1

(Table 1), with the aim to occupy available space observed in

the structure and increase van der Waals interactions (for

example with Gly132) for improved potency. Satisfyingly

triﬂuoromethylpyridine 7 (K_i= 0.007 μM, p-JNK IC₅₀= 0.183

μM) and cyanopyridine 8 (K_i= 0.022 μM, p-JNK IC₅₀= 0.188

μM) showed improved biochemical potency and activity in a

cell-based assay measuring levels of JNK phosphorylation (p-

JNK) induced by DLK overexpression.¹³These results

validated our strategy for the pyrazole core replacement.

Consistently, the more polar cyanopyridine 8 (LipE 4.1)

showed the best lipophilic eﬃciency. In a MDCK-MDR1 assay,

3 (BA/AB = 36) and 8 (BA/AB = 95) are eﬄux substrates

despite low tPSA (<80 Å²) due to a basic amine. Holding the

hinge binder constant to the more lipophilic eﬃcient 2-

aminoisonicotinonitrile moiety, we then investigated capping

the piperidine N4 to reduce the hydrogen-bond donor count

and basicity in order to improve permeability. Small capping

groups such as acyl and oxetane, compounds 9 (K_i= 0.44 μM,

MDRI-MDCK BA/AB = 9.1) and 10 (K_i= 0.549 μM, MDRI-

MDCK BA/AB 1.9), respectively, showed that the oxetane

moiety improved eﬄux liability.¹³Combining this result with

the pyrazole N1 cyclopentyl side chain from compound 8 gave

us 11, where a desirable balance of potency and eﬄux

Table 1. ROCS Shape-Overlay Analysis

a

To assess core replacements, a model based on the subunit 2,6-

dimethyl-N-(4-(triﬂuoromethyl)pyridin-2-yl)pyrimidin-4-amine bioac-

tive conformation was obtained by generation of the lowest-energy

conformation for ﬁve-membered heterocyclic systems (pyrazole,

thiazole, or imidazole) with OMEGA and subsequent quantum

mechanical minimization at the B3LYP/6-31G** level of theory (see

Supporting Information for computational methods). The three

heterocyclic replacements evaluating favorably through this analysis,

N-(1,5-dimethyl-1H-pyrazol-3-yl)-4-(triﬂuoromethyl)pyridin-2-amine,

4,5-dimethyl-N-(4-(triﬂuoromethyl)pyridin-2-yl)thiazol-2-amine, and

N-(1,2-dimethyl-1H-imidazol-4-yl)-4-(triﬂuoromethyl)pyridin-2-

amine were scored using a ROCS shape-overlay aiming to retain global

shape similarity with respect to elaborated compound 1. Expansion

with the proposed P-loop and solvent side chains showed a suitable

level of shape preservation with respect to compound 1 and similar

TanimotoShape and TanimotoCombo scoring between the three

heterocyclic systems.

properties was realized (K_i= 0.042 μM and p-JNK IC₅₀

=

→ cJun) and DLK homologues (MLK 1/2/3) was a

prerequisite to in vivo evaluation.²⁷

0.536 μM, MDR1-MDCK BA/AB 1.4, AB 8.8 × 10⁻⁶cm/s).

Our crystal structure of 11 bound to DLK supported these

designs (Figure 4). The core of the scaﬀold is very closely

matched to 3, with consistent position of the protein features

and core-interacting side chains. The cyano group extends into

the back region near residues Lys152, Val139, and Gln136. The

introduced cyclopentyl group maintains a similar DLK P-loop

conformation as observed with 3, but the greater bulk causes a

small additional shift upward and outward. The distal oxetane

capping group extends, as expected, away from the protein into

the less constrained solvent-accessible environment, permitting

more facile modulation of ligand properties. Based on these

results we looked to improve potency by further extending the

pyrazole N1 SAR under the P-loop and holding the 2-((5-(1-

(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-yl)amino)-

isonicotinonitrile subunit constant (Table 3). The pyrazole N1-

(3,3-diﬂuorocyclopentyl) analog 12 showed comparable

biochemical and in vitro microsomal data to 11 but diminished

cellular potency (p-JNK IC₅₀= 0.721 μM) and permeability

(MDR1-MDCK BA/AB 5.8). Polarity under the P-loop was

not well-tolerated. 1-(Tetrahydrofuran-3-yl) 13 resulted in a

loss of biochemical potency with increased eﬄux liability (K_i=

0.378 μM, MDR1-MDCK BA/AB 7.6). Similarly ring

expansion to 1-(tetrahydro-2H-pyran-4-yl) 14 showed a loss

in potency and high eﬄux liability (K_i= 0.593 μM, MDR1-

MDCK BA/AB 15). Extending under the P-loop with 1-

Starting with 1-isopropyl-5-(piperidin-4-yl)-1H-pyrazol-3-

amine, we evaluated the C4 and C5 position of the

aminopyridine hinge binder. Similar to the reported SAR of

the pyrimidine series (1),¹³the 2-aminopyridine C4 sub-

stitution was optimal for potency (matched pairs compound 3,

K_i= 0.145 μM/compound 5, K_i= >1.6 μM and compound 4,

K_i= 0.160 μM/compound 6, K_i= >1.6 μM) (Table 2).

Furthermore, comparison of lipophilic eﬃciencies demonstra-

ted that the 2-amino-4-cyanopyridine 4 (LipE 3.9) was superior

to 2-amino-4-triﬂuoromethylpyridine 3 (LipE 2.6). Our X-ray

structure of DLK complexed with triﬂuoromethylpyridine 3

conﬁrmed that the binding mode is consistent with the

calculated low-energy ligand conformation (see Supporting

Information Figure S2 and Table S2) for the pyrazole group

replacement (Figure 3). The central pyrazole core and pyridine

ring lie on the platform of the C lobe Gly196 and Leu243, with

hydrogen bonds formed between the ligand and backbone

carbonyl and amide atoms of the hinge residue Cys193. In

addition, the pyridine C6−H contributes to a noncanonical

hydrogen bond with the Glu191 backbone carbonyl. The core

pyrazole ring is also aligned to participate in π orbital

interactions with the side chain of Phe192, and the piperidine

moiety extends to the front solvent-exposed opening. In

contrast to the apo example, the activation segment is

disordered in our ligand-bound structures (residues 257−

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a

Table 2. Pyrazole N1 and C5-Substituent and Pyridine C4/C5-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. LipE = −log K_i

− clogP.

(cyclobutylmethyl) 15 retained potency (K_i= 0.133 μM, p-JNK

IC₅₀= 1.4 μM) but not signiﬁcantly. Finally we investigated

SAR in the solvent exposed region by replacing N-

oxetanylpiperidine 11 with N-oxetanylazetidine 16, N-oxeta-

nylpyrrolidine 17, and furanyl 18. In each case comparable

biochemical and cellular potency to 11 was observed.

Compounds 16 (MDRI-MDCK BA/AB 3.3, tPSA 79 Å²)

and 17 (MDR1-MDCK BA/AB 3.7, tPSA 79 Å²) suﬀered from

potential eﬄux liability. Compound 18 (MDR1-MDCK BA/AB

1) showed improved eﬄux liability but at the expense of

metabolic stability in liver microsomes. Finally we evaluated the

thiazole and imidazole core replacements outlined in Table 1.

To reduce lipophilicity of the thiazole scaﬀold, the proposed

cyclopentyl group (Table 1, clogP 5.3) was substituted with a

cyclopropyl side-chain (clogP 4.5). Additionally, for both the

thiazole and imidazole core replacements we replaced the 4-

(triﬂuoromethyl)pyridin-2-amine with the 2-aminoisonicotino-

nitrile hinge binder and N-acetylpiperidine with the more

favorable N-oxetanylpiperidine solvent exposed side-chain

(TanimotoShape/TanimotoCombo 0.777/1.458 and 0.803/

1.371, respectively, Table 4). Although the TanimotoShape and

TanimotoCombo scoring were comparable between the ﬁve-

membered heterocyclic replacements highlighted in Table 1

(pyrazole 11 [TanimotoShape/TanimotoCombo 0.819/1.509],

thiazole 19 and imidazole 20), the computational method

correctly rank ordered the suitability of these core changes

(pyrazole 11 K_i= 0.042 μM > thiazole 19 K_i= 0.569 μM ≫

imidazole 20 K_i> 1.6 μM).

We compared the crystal structures of pyrazole 11 to that of

DLK bound to pyridine 2 (Approach 1). The exact

superposition of the 2-amino-4-cyanopyridine hinge binder of

the inhibitors and the close alignment of the solvent exposed

N-oxetanylpiperidine moieties is a testament to a successful

design strategy incorporating the altered vectors emanating

from the ﬁve- versus six-membered core ring diﬀerences

(Figure 5). Both ligands have relatively large chemical moieties

positioned under the P-loop resulting in very similar protein

backbone conformations. Compound 2 positions a 3,3-

diﬂuoropyrrolidine (compared to cyclopentyl 11) group that

is likely connected to the upward-turned position of Gln136

relative to downward wrap around the ligand with 11 (Figure

5b), which in turn induces a small shift in the Asp254 rotamer.

These minor adjustments illustrate that the DLK P-loop

includes subtle as well as coarser conformational shifts in

response to the diﬀerent P-loop-oriented groups of the two

ligands. These nuanced considerations underpin the well-

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of 58 representative kinases revealed good overall selectivity

with no oﬀ-target kinases displaying >70% inhibition @ 1 μM

(Figure 6). We then further evaluated 11 against other

members of the mixed lineage kinase family and kinases within

the JNK pathway through a 10-point dose−response analysis in

an enzymatic assay. Good selectivity was observed against all

JNK isoforms (Table 5, JNK1 IC₅₀= 1.04 μM, JNK2 IC₅₀= 5

μM, and JNK3 IC₅₀= 2.1 μM), and no activity was observed at

the top concentrations tested for MKK4/7 and MLKs (Table

5). To conﬁrm that inhibitor 11 suﬃciently inhibited DLK

activity to elicit protection of neurons from degeneration, it was

tested in a high content neurodegeneration assay where it

protected neurons with an EC₅₀of 2.15 0.56 μM.²⁸As

expected, the EC₅₀required for protection of neurons was

higher than that observed in the p-JNK assay, as near complete

inhibition of DLK activity is required to elicit neuroprotection

in this setup.¹⁰This is consistent with the ratio of the cell-based

activity/neuroprotection EC₅₀observed in the same assay for

JNK published inhibitor SP600125.²⁸To conﬁrm that the

neuroprotection observed with 11 was a result selective DLK

inhibition rather than inhibition of downstream kinases, levels

of p-MKK4, p-MKK7, p-JNK, and p-c-Jun were examined

following overexpression of DLK or constitutively active JNK

construct (CA-JNK) to induce pathway activity.²⁹Over-

expression of DLK resulted in increases in the phosphorylation

of MKK4, MKK7, JNK, and c-Jun, while CA-JNK only induced

JNK and c-Jun, consistent with previous studies.^5,29The

addition of 11 reduced phosphorylation of all of these signaling

components following DLK overexpression, indicating that

inhibition of pathway activity indeed occurred at the level of

DLK rather than a downstream signaling component (Figure

7). This was further supported by the lack of pathway inhibition

observed with 11 following overexpression of CA-JNK (Figure

7). Levels of total JNK were not aﬀected by compound

treatment. Taken together, the overall properties of inhibitor 11

were suitable to warrant progression of this molecule for in vivo

assessment.

Figure 3. Crystal structure of compound 3 bound to DLK. Ligand

carbon atoms are colored green to distinguish it from the surrounding

ligand-proximal side chains (brown) and ordered water molecules

(cyan). Hydrogen-bond interactions (dashed lines) between the ligand

and the DLK hinge region anchor the core scaﬀold pyridine and

pyrazole ring systems, presenting the triﬂuoromethyl moiety near the

gatekeeper Met190. The malleable DLK P-loop curls around the

ligand isopropyl with a notable inward orientation of Gln136 at its tip.

The foreground piperidine moiety clears the lower hinge segment and

extends from the binding cavity toward greater solvent exposure.

Inhibitor 11 exhibited moderate pharmacokinetic properties

and good brain penetration in rat (CL_p= 34 mL/min/kg, V_dss

=

4.4 L/kg, t_1/2= 1.7 h, F = 58%, B_u/P_u= 0.354). Although

having similar total plasma clearance to inhibitor 2 (Table 6),

inhibitor 11 aﬀorded signiﬁcantly improved free clearance (CL_u

= 327 mL/min/kg). Additionally, 11 had suﬃcient oral

exposure in mouse (50 mpk C_max= 5.9 μM, T_max= 0.25 h,

AUC_0−6h= 15.8 h*μM, free brain @ 6 h = 0.050 μM) to

warrant examination in a PK/PD model based on the optic

nerve crush injury in mice. As previously described, this model

mimics the neuronal degeneration that occurs in glaucoma or

optic neuropathy.^9,10Optic nerve crush robustly induces

phosphorylation of c-Jun (p-c-Jun) in a DLK-/JNK-dependent

fashion and could thus be used as a pharmacodynamic readout

of DLK inhibition in vivo.^9,10,13Animals were dosed orally with

either inhibitor 11 or vehicle control 30 min prior to nerve

crush injury, and levels of p-c-Jun in retina were measured 6 h

after injury. As predicted by the levels of free compound in

brain, treatment with 75 mg/kg inhibitor 11 resulted in a

reduction of p-c-Jun present in retina comparable to inhibitor 2

(Figure 8).¹³

Figure 4. Crystal structure of compound 11 bound to DLK.

Representations are similar to Figure 3, here with ligand carbon

atoms colored orange. The core scaﬀold maintains key hydrogen-bond

interactions with the hinge segment, while the back region cyano

group and the forward cyclopentyl moiety engage the P-loop. The

solvent-exposed portion of the ligand extends an oxetane beyond the

piperidine.

aligned structural features that are achieved around the pyridine

to pyrazole core scaﬀold hopping design.

From the SAR examined, inhibitor 11 oﬀered the best

balance for potency, in vitro metabolic stability and desirable

eﬄux properties. Broad proﬁling in an Life Technologies panel

CHEMISTRY

■

The syntheses of 3−6, 9, 10, 15, and 16 were carried out as

outlined in Scheme 1. The synthesis of tert-butyl 4-(3-amino-1-

isopropyl-1H-pyrazol-5-yl)piperidine-1-carboxylate (38) com-

E

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a

Table 3. Pyrazole N1-Substituent SAR

a

Compounds 12, 13, 17, and 18 were tested as racemates, and also subsequent separation into the chirally pure enantiomers showed no additional

beneﬁt (not reported). All assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced

b

results within 3-fold of the reported mean. MDCK-MDR1 human P-gp transfected cell line. Basolateral-to-apical/apical-to-basolateral. Units = ×

c

d

e

10⁻⁶cm s⁻¹. Liver microsome-predicted hepatic clearance. H/R/M = human/rat/mouse. LipE = −log K_i− clogP.

a

Table 4. Thiazole and Imidazole Core Examples

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. LipE = −log K_i− clogP.

menced with the acylation of acetonitrile anion with

commercially available 1-tert-butyl 4-methylpiperidine-1,4-

dicarboxylate (CH₃CN, K^tOBu, THF) to provide β-ketonitrile

25. Condensation of the β-ketonitrile 25 with hydrazine

produced aminopyrazole 29 which subsequently aﬀorded tert-

butyl 4-(3-(1,3-dioxoisoindolin-2-yl)-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (33) upon treatment with phthalic

anhydride. N-pyrazole alkylation with 2-iodopropane (Cs₂CO₃,

DMF), followed by protecting group removal, produced tert-

butyl 4-(3-amino-1-isopropyl-1H-pyrazol-5-yl)piperidine-1-car-

F

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Figure 5. Comparison of crystal structure binding modes for compounds 11 and 2 to DLK. The initial lead pyridine-core 2 is shown with carbon

atoms in navy blue and DLK protein in ivory, while the scaﬀold-hop pyrazole-core 11 is depicted with carbon atoms in orange and DLK protein in

white. (a) DLK binding mode showing the drape of the P-loop (upper right) around the ligand. The two compounds have an almost exact

superposition of the shared back region cyano pyridine moiety. The slightly larger diﬂuoro-pyrrolidine of 2 engages the P-loop to create a slightly

outward trajectory (ivory). (b) A cut-away top-down view of the ligand with the DLK hinge at upper left shows the precise cyano-pyridine alignment

and conserved hinge interactions of both ligands. The ﬁve-membered pyrazole versus six-membered pyridine core ring provides altered vectors,

though the outer substituents converge into shared spatial position. The shifted orientation of Gln136 seen at right from its inward ligand-proximal

juxtaposition in the compound 11 structure (orange/white) to an outward display in the case of compound 2 binding (navy/ivory) is likely related to

the shifted P-loop response from the diﬂuoro-pyrrolidine moeity.

Figure 6. Kinase selectivity of compound 11 @ 1uM (24 × DLK K_i). Values are reported as percent inhibition and determined at Life Technologies.

58 representative kinases were selected from various branches of the kinome tree that were frequent oﬀ-targets against a large and diverse panel of

internally derived inhibitors. Kinases with >50% inhibition are CSF1R (IC₅₀= 0.057 mM), Flt3 (IC₅₀= 0.322 mM), GS3_beta (IC₅₀= 0.682 mM),

Kit (IC₅₀= 0.534 mM), Src (IC₅₀= 1.01 mM), TrkA (IC₅₀= 0.464 mM), and TrkB (IC₅₀= 1.19 mM). Biochemical IC₅₀values were determined at

Life Technologies.

a

Table 5. Kinase Selectivity of Compound 11

K_i(μM)

DLK

IC₅₀(μM)

b

MKK4

>5

MKK7

> 5

JNK1

1.04

JNK2

5

JNK3

2.1

MLK1

>10

MLK2

>10

MLK3

>10

0.042

a

b

Biochemical IC₅₀determination. Biochemical IC₅₀determination at Life Technologies.

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

required heteroaryl bromides followed by N-Boc deprotection

furnished 3−6. Acylation of compound 4 with acetic anhydride

provided acetamide 9, while reductive amination with oxetan-3-

G

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Figure 8. Compound 11 displays comparable activity to compound 2

in the optic nerve crush model. Levels of p-c-Jun in retinal lysates

measured by ELISA following nerve crush and treatment with

inhibitor 11 or 2. Values are presented relative to uncrushed vehicle

controls, **p < 0.01. Bars represent the mean and error bars represent

SEM. Dots represent data points from individual animals in the study.

Figure 7. Transfection of HEK293 cells with Flag DLK results in

increase phosphorylation of MKK4 (p-MKK4), MKK7 (p-MKK7),

JNK (p-JNK), and c-Jun (p-c-Jun), while transfection of a kinase dead

form of DLK (Flag DLK K152A) does not. Addition of increasing

concentrations of compound 11 (0.1, 1, or 10 μM) reduces levels p-

MKK4, p-MKK7, p-JNK, and p-c-Jun. Levels of total JNK are not

aﬀected by addition of compound. Ten μM of compound 11 is not

able to reduce p-c-Jun induced by overexpression of a constitutively

active JNK (Myc CA-JNK).

pyrazol-5-yl)piperidine-1-carboxylate (45). Buchwald−Hartwig

reaction of 45 with 2-bromo-4-(triﬂuoromethyl)pyridine and 2-

bromoisonicotinonitrile provided compounds 7 and 8,

respectively, after N-Boc deprotection. Oxetane 11 was

obtained by reductive amination with oxetan-3-one. The

syntheses of 12−14, 17, and 18 were conducted as outlined

in Scheme 3. Sandmeyer reaction of tert-butyl 4-(3-amino-1H-

pyrazol-5-yl)piperidine-1-carboxylate (29) resulted in iodopyr-

azole 46 which was utilized in the syntheses of intermediates

50−52. Hafnium chloride³⁰catalyzed conjugate addition of 46

to cyclopent-2-enone followed by ﬂuorination with DAST

aﬀorded tert-butyl 4-(1-(3,3-diﬂuorocyclopentyl)-3-iodo-1H-

pyrazol-5-yl)piperidine-1-carboxylate (50). Intermediates 51

and 52 were obtained by the reaction of 46 with

tetrahydrofuran-3-yl methanesulfonate and tetrahydro-2H-

pyran-4-yl methanesulfonate respectively (NaH, DMF). Ami-

nopyrazoles 31 and 32 followed the same procedure as 29

(Scheme 1) with substitution of tert-butyl 3-methyl pyrrolidine-

1,3-dicarboxylate (23) and methyl tetrahydrofuran-3-carbox-

ylate (24) for 1-tert-butyl 4-methylpiperidine-1,4-dicarboxylate,

respectively. Subsequent iodination and N-pyrazole alkylation

with bromocyclopentane yielded intermediates 53 and 54.

Modiﬁed Goldberg coupling³¹⁻³³of 50−53 with amino-

isonicotinonitrile, acid-mediated N-Boc deprotection, and

reductive amination with oxetan-3-one furnished 12−14 and

17. Compound 18 was obtained via Goldberg coupling of

aminoisonicotinonitrile with 54. The preparation of compound

19 was performed as shown in Scheme 4. Starting with

commercially available 4-cyclopropylthiazol-2-amine (55)

mono N-Boc protection (56) followed by chlorination (N-

chlorosuccinimide) led to intermediate 57. Suzuki−Miyaura

cross coupling of 57 with tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate pro-

vided alkene 58 which was reduced by hydrogenation to obtain

intermediate 59. Removal of the tert-butoxycarbonyl protective

groups provided 60, and subsequent reductive amination with

oxetan-3-one provided the N-oxetanylpiperidine 61. Com-

pound 19 was attained via Buchwald−Hartwig coupling of

chloroisonicotinonitrile with 61. The synthesis of compound

20 was performed as highlighted in Scheme 5. Iodination of

commercially available 2-cyclopentylimidazole (62) followed by

regioselective reduction gave monoiodoimidazole 64.³⁴Inter-

a

Table 6. PK Properties of DLK Inhibitors 1, 2, and 11

1

2

11

b

Rat

CL_p(mL min⁻¹kg⁻¹

CL_u(mL min⁻¹kg⁻¹

)

45

45000

3.8

30

2500

3.7

34

327

4.4

c

V_dss(L kg⁻¹

)

t_1/2(h)

1.2

1.8

1.7

F (%)

16

63

58

d

B_u/P_u

1.3

0.7

0.35

0.03

Free AUC/dose (h·kg·L⁻¹

)

e

0.00006

0.004

Mouse (PO 50 mg kg⁻¹

C_max(μM)

)

4.38

0.8

5.9

0.25

15.8

0.050

T_max(h)

AUC_0−6h(h·μM)

B_u(μM) @ 6 h

12.8

0.004

a

Rat PK properties of compounds 1, 2, and 11. Mouse 50 mg kg⁻¹PO

b

PK properties of compounds 2 and 11. Compounds were dosed iv

(0.4 or 1 mg kg⁻¹) as a 60% PEG solution and po (1 or 5 mg kg⁻¹) as

c

an aqueous suspension with 1% methylcellulose. CL_u= CL/f_u(f_u=

d

fraction unbound in plasma). Unbound brain/unbound plasma AUC

e

ratio (unless noted otherwise). Compounds dosed po (50 mg kg⁻¹)

as an aqueous suspension with 1% methylcellulose.

one provided N-oxetane 10. Similar to the synthesis of 10,

compound 15 was obtained by alkylation of 33 with

(bromomethyl)cyclobutane. Likewise, compound 16 was

made with commercially available 1-tert-butyl 3-methyl

azetidine-1,3-dicarboxylate (22) with subsequent alkylation of

34 with bromocyclopentane. The preparation of compounds 7,

8, and 11 was performed as shown in Scheme 2. Protection of

tert-butyl 4-(3-amino-1H-pyrazol-5-yl)piperidine-1-carboxylate

(29) with 2,5-hexanedione aﬀorded tert-butyl 4-(3-(2,5-

dimethyl-1H-pyrrol-1-yl)-1H-pyrazol-5-yl)piperidine-1-carbox-

ylate (43). N-pyrazole alkylation with iodocyclopentane

followed by 2,5-dimethylpyrrole deprotection (NH₂OH,

KOH, EtOH) yielded tert-butyl 4-(3-amino-1-cyclopentyl-1H-

H

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a

Scheme 1

a

Reagents and conditions: (a) CH₃CN, K^tOBu, THF, 23 °C, 1 h; (b) hydrazine monohydrate, i-PrOH, 80 °C, 18 h; (c) phthalic anhydride, 1,4-

dioxane, 90 °C, 18 h; (d) R-I or R-Br, Cs₂CO₃, DMF, or DMA, 50−110 °C, 3 h−16 h; (e) hydrazine hydrate, MeOH, 23 °C, 30 min; (f) i. R-Br,

Pd₂(dba)₃, Xantphos, t-BuONa, 1,4-dioxane, 80 °C, 3h; ii. HCl in 1,4-dioxane, MeOH, 23 °C, 16 h; (g) acetic anhydride, TEA, DMF, 40 °C, 16 h,

58%; (h) 3-oxetanone, Na(OAc)₃BH, DCM, 23 °C, 1 h.

mediate 65 was obtained by reaction of 64 with tert-butyl 4-

((methylsulfonyl)oxy)piperidine-1-carboxylate (NaH, DMF).

Goldberg coupling of 65 with aminoisonicotinonitrile followed

by acid-mediated N-Boc deprotection and ﬁnal reductive

amination with oxetan-3-one procured analog 20.

potential for further optimization as a small molecule

therapeutic.

METHODS

■

DLK Biochemical Assay. The biochemical assay was performed as

previously described.³⁵

p-JNK Cell Assay. The p-JNK cell assay for generation of IC₅₀

CONCLUSIONS

values was performed as previously described.¹³

■

The scaﬀold-hopping approach described herein, facilitated by

our determination of the ﬁrst DLK structures, provided a

complementary method to optimize compound 1 by improving

physiochemical properties. Although less potent than the

inhibitor 2,¹³inhibitor 11 possessed improved free drug

exposure; thus providing inhibitors with equivalent in vivo

inhibition of the DLK/JNK pathway, but with increased

DLK and CA-JNK Transfection Cell Assays. DLK and CA-JNK

transfection assays to assess pathway selectivity were performed as

described.⁵Cells were treated with compound for 5 h, and the

transfection was performed 24 h prior to compound treatment.

In Vitro Axon Degeneration Cell Assay. Assay was conducted as

previous described.³⁶

MKK4 and MKK7 Biochemical Assays. MKK4 enzymatic

reaction was initiated by combining 30 nM MKK4 with 100 μM

I

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a

Scheme 2

a

Reagents and conditions: (a) 2,5-hexadione, MgSO₄, toluene, 40 °C, 16 h, 97% crude yield; (b) iodocyclopentane, Cs₂CO₃, DMF, 50 °C, 16 h, 69%

yield; (c) hydroxylamine, KOH, EtOH, 80 °C, 16 h, 34% yield; (d) i. R-Br, Pd₂(dba)₃, Xantphos, t-BuONa, 1,4-dioxane, 80 °C, 3h; ii. HCl in 1,4-

dioxane, MeOH, 23 °C, 16 h; (e) 3-oxetanone, Na(OAc)₃BH, DCM, 23 °C, 1 h.

a

Scheme 3

a

Reagents and conditions: (a) NaI, NaNO₂, p-toluenesulfonic acid monohydrate, ACN, H₂O, 0 °C, 1 h; (b) cyclopent-2-eneone, hafnium

tetrachloride, DCM, 23 °C, 12 h, 100% crude yield; (c) R-OSO₂Me, NaH, DMF, 60−90 °C, 2−4 h; (d) bromocyclopentane, Cs₂CO₃or K₂CO₃,

DMF, 50−80 °C, 2−3 h; (e) DAST, DCM, 23 °C, 12 h, 20% yield; (f) i. 2-aminoisonicotinonitrile, CuI, N¹,N²- dimethylcyclohexane-1,2-diamine,

K₃PO₄, DMF, 160 °C microwave, 1 h; ii. TFA, DCM, 23 °C, 3 h; iii. 3-oxetanone, Na(OAc)₃BH, DCM, 23−60 °C, 1 h.

peptide substrate (KFMMTPpYVVTR), 5 μM ATP, and 3.3 nM

ﬂuorescence labeled phospho-peptide probe (MpTPpYV) in buﬀer

containing 50 mM HEPES pH 7.5 0.1% Pluronic F-127, 5 mM MgCl₂,

10 mM DTT, 6.25 mM NaCl, 0.1 mM EGTA, and 0.01% BSA. The

reaction mixture was incubated at 23 °C for 1 h before the addition of

stop mixture (50 mM HEPES pH 7.5 0,1% Pluronic F-127, 6.25 mM

NaCl 0.1 mM EGTA, 22.5 mM EDTA, 0.01% BSA and P-SAPK/JNK

(T183/Y185) (81E11) Rabbit mAb in 1:1000 dilution). The

ﬂuorescence polarization (FP) of the competitive phospho-peptide

probe in the ﬁnal mixture was detected at excitation wavelength of 633

nm and emission wavelength of 670 nm using FCS+ Insight reader

(Evotec AG, Germany). The enzymatic activity in the presence of

inhibitor was normalized against the signal of positive control. MKK7

reaction was carried out with 100 μM peptide substrate

(KFMMpTPYVVTR) and 30 μM ATP under the same reaction

condition.

the K_ifor the compound against human recombinant DLK in the DLK

enzyme assay. The kinase assays were carried out using Z′-LYTE

Technology (Life Technologies, Madison WI) which measured labeled

peptide phosphorylation via ﬂuorescence resonance energy transfer

(FRET) following protocols developed and performed by Life

Technologies.³⁷

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, C57/Bl6 mice were dosed with compound 11

orally as an MCT suspension. Optic nerve crush studies were

conducted as described,⁹except p-c-Jun was measured at 6 h by MSD

ELISA (Mesoscale Discovery).

EXPERIMENTAL SECTION

■

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 chromatography was performed with Isco

In Vitro Transporter Assays. The in vitro transporter assays were

performed as previously described.¹³

Life Technologies Kinase Assays. Compound 11 was tested in

Life Technologies’ SelectScreen (Madison, WI) against 58 representa-

tive kinases at a concentration of 1 uM, which is 24-fold greater than

J

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a

Scheme 4

a

Reagents and conditions: (a) di-tert-butyl dicarbonate, 80 °C, 5 h, 74% yield; (b) N-chlorosuccinimide, 0 °C, 2 h, 85% yield; (c) tert-butyl 4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate, Pd₂(dba)₃, Xantphos, 1,4-dioxane, H₂O, 100 °C, 4 h, 47%

yield; (d) H₂, MeOH, 10% Pd/C, 55 psi, 98% crude yield; (e) TFA, DCM, 30 °C, 4 h, 100% crude yield; (f) 3-oxetanone, Na(OAc)₃BH, DCM, 23

°C, 1 h, 62% crude yield; (g) 2-chloroisonicotinonitrile, Pd(t-Bu₃P)₂, K₃PO₄, 1,4-dioxane, 100 °C, 3 h, 5% yield.

a

instrument with an Ultimate C-18 (3.0 × 50 mm; 3 μm particle

size) stationary phase, and a gradient of water/acetonitrile (10−80%

over 6 min 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.

Scheme 5

N-(1-Isopropyl-5-(piperidin-4-yl)-1H-pyrazol-3-yl)-4-

(triﬂuoromethyl)pyridin-2-amine (3). A mixture of 38 (0.10 g, 0.33

mmol), 2-bromo-4-(triﬂuoromethyl)pyridine (59 mg, 0.26 mmol),

sodium tert-butoxide (32 mg, 0.33 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (16 mg, 0.017 mmol),

and tris(dibenzylideneacetone)dipalladium(0) (15 mg, 0.026 mmol)

in 1,4-dioxane (10 mL) was heated at 80 °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 residue was dissolved in

methanol (2 mL), and a 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 and puriﬁed by preparative

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

a

Reagents and conditions: (a) I₂, aq NaOH, 50 °C, 6 h, 35% yield; (b)

Na₂SO₃, EtOH, reﬂux, 10 h, 74% yield; (c) tert-butyl 4-

((methylsulfonyl)oxy)piperidine-1-carboxylate, NaH, DMF, 100 °C,

8 h, 8% yield; (d) i. 2-aminoisonicotinonitrile, CuI, N¹,N²-

dimethylcyclohexane-1,2-diamine, K₃PO₄, DMF, 160 °C microwave,

1 h; ii. HCl in1,4-dioxane, MeOH, 23 °C, 16 h; iii. 3-oxetanone,

Na(OAc)₃BH, DCM, 23 °C, 1 h, 8% yield.

1

(64 mg, 55% yield). LCMS: m/z = 354.1 [M + H]⁺; H NMR (400

MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.46−8.24 (m, 2H), 7.61 (s, 1H),

7.00−6.87 (m, 1H), 6.12 (s, 1H), 4.62−4.43 (m, 1H), 3.17 (d, J = 12.4

Hz, 2H), 3.01−2.87 (m, 1H), 2.87−2.73 (m, 2H), 1.86 (d, J = 13.1

Hz, 2H), 1.70−1.53 (m, 2H), 1.37 (d, J = 6.4 Hz, 6H).

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

× 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

Bruker 300, 400, or 500 MHz spectrometers, 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 × 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

2-(1-Isopropyl-5-(piperidin-4-yl)-1H-pyrazol-3-ylamino)-

isonicotinonitrile (4). A mixture of 38 (821 mg, 2.00 mmol), 2-

bromoisonicotinonitrile (403 mg, 2.20 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (89.0 mg, 0.150 mmol),

tris(dibenzylideneacetone)dipalladium(0) (92.0 mg, 0.100 mmol), and

sodium tert-butoxide (269 mg, 2.80 mmol) in 1,4-dioxane (8 mL) was

heated at 80 °C for 16 h. The reaction mixture was ﬁltered and diluted

with water (15 mL). The resulting solution was extracted with ethyl

acetate (2 × 30 mL). The collected organic was dried over anhydrous

sodium sulfate, ﬁltered, and concentrated under reduced pressure. The

residue was dissolved in dichloromethane (15 mL), and a 4.0 M

solution of hydrogen chloride in 1,4-dioxane (0.80 mL, 3.20 mmol)

was added. After 16 h at 23 °C, the reaction mixture was concentrated

under reduced pressure to aﬀord crude title compound (597 mg, 66%

crude yield). A portion of the crude (102 mg, 0.330 mmol) was

puriﬁed by preparative reverse-phase HPLC to aﬀord the title

compound as an oﬀ-white solid (22 mg, 21%). LCMS: m/z = 311.1

[M + H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.75 (s, 1H), 8.31 (d, J

= 5.1 Hz, 1H), 7.50 (s, 1H), 7.01 (d, J = 5.1 Hz, 1H), 6.13 (s, 1H),

4.58−4.39 (m, 1H), 3.02−2.91 (m, 2H), 2.84−2.69 (m, 1H), 2.69−

K

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2.54 (m, 1H), 2.07 (s, 1H), 1.79−1.67 (m, 3H), 1.51−1.41 (m, 2H),

1.36 (d, J = 6.4 Hz, 6H).

methane (15 mL) and saturated aqueous ammonium chloride solution

(15 mL). The organic was separated, and the aqueous layer was further

extracted with dichloromethane (2 × 5 mL). The collected organic

was dried over anhydrous sodium sulfate, ﬁltered, and concentrated

under reduced pressure. Puriﬁcation by preparative reverse-phase

HPLC aﬀorded the title compound as a white solid (82 mg, 64%).

N-(1-Isopropyl-5-(piperidin-4-yl)-1H-pyrazol-3-yl)-5-

(triﬂuoromethyl)pyridin-2-amine (5). The title compound (17 mg,

15% yield) was prepared in a manner analogous to 3 by substituting 2-

bromo-5-(triﬂuoromethyl)pyridine for 2-bromo-4-(triﬂuoromethyl)-

pyridine. LCMS: m/z = 354.1 [M + H]⁺; ¹H NMR (400 MHz,

DMSO-d₆) δ 9.90 (s, 1H), 8.44 (s, 1H), 7.86−7.78 (m, 1H), 7.25 (d, J

= 8.9 Hz, 1H), 6.21 (s, 1H), 4.55−4.43 (m, 1H), 2.98 (d, J = 12.2 Hz,

2H), 2.82−2.70 (m, 1H), 2.64−2.53 (m, 2H), 2.10 (s, 1H), 1.77−1.68

(m, 2H), 1.51−1.33 (m, 8H).

1

LCMS: m/z = 367.2 [M + H]⁺; H NMR (400 MHz, DMSO-d₆) δ

9.76 (s, 1H), 8.32 (d, J = 5.1 Hz, 1H), 7.51 (s, 1H), 7.05−6.98 (m,

1H), 6.16 (s, 1H), 4.58−4.40 (m, 5H), 3.47−3.35 (m, 1H), 2.82−2.65

(m, 3H), 1.95−1.74 (m, 4H), 1.66−1.51 (m, 2H), 1.36 (d, J = 6.4 Hz,

6H).

6-(1-Isopropyl-5-(piperidin-4-yl)-1H-pyrazol-3-ylamino)-

nicotinonitrile (6). The title compound (29 mg, 28% yield) was

prepared in a manner analogous to 3 by substituting 6-

bromonicotinonitrile for 2-bromo-4-(triﬂuoromethyl)pyridine.

2-((1-Cyclopentyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-

yl)amino)isonicotinonitrile (11). A mixture of 8 (236 mg, 0.600

mmol) and oxetan-3-one (87.0 mg, 1.20 mmol) in dichloromethane (5

mL) was stirred at 23 °C for 15 min. Sodium triacetoxyborohydride

(381 mg, 1.80 mmol) was added, and the mixture was stirred at 23 °C

for an additional 1 h. The reaction mixture was partitioned between

dichloromethane (15 mL) and saturated aqueous ammonium chloride

solution (15 mL). The organic was separated, and the aqueous layer

was further extracted with dichloromethane (2 × 5 mL). The collected

organic was dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. Puriﬁcation by preparative

reverse-phase HPLC aﬀorded the title compound as a white solid (142

1

LCMS: m/z = 311.3 [M + H]⁺; H NMR (400 MHz, DMSO-d₆) δ

10.11 (s, 1H), 8.53 (s, 1H), 7.87 (dd, J = 8.8, 2.3 Hz, 1H), 7.20 (s,

1H), 6.21 (s, 1H), 4.50 (p, J = 6.4 Hz, 1H), 3.02−2.93 (m, 2H), 2.82−

2.71 (m, 1H), 2.58 (t, J = 12.1 Hz, 2H), 2.05 (s, 1H), 1.77−1.68 (m,

2H), 1.50−1.38 (m, 2H), 1.36 (d, J = 6.4 Hz, 6H).

N-(1-Cyclopentyl-5-(piperidin-4-yl)-1H-pyrazol-3-yl)-4-

(triﬂuoromethyl)pyridin-2-amine (7). The title compound (5.4 mg,

7% yield) was prepared in a manner analogous to 3 by substituting

tert-butyl 4-(3-amino-1-cyclopentyl-1H-pyrazol-5-yl)piperidine-1-car-

boxylate for tert-butyl 4-(3-amino-1-isopropyl-1H-pyrazol-5-yl)-

1

mg, 60%). H NMR (500 MHz, DMSO-d₆) δ 9.79 (s, 1H), 8.33 (d, J

= 5.1 Hz, 1H), 7.58 (zs, 1H), 7.03 (dd, J = 5.1, 1.4 Hz, 1H), 6.14 (s,

1H), 4.69 (m,z 1H), 4.55 (dd, J = 6.3, 6.3 Hz, 2H), 4.44 (dd, J = 6.3,

6.3 Hz, 2H), 3.40 (p, J = 6.3 Hz, 1H), 2.80−2.70 (m, 3H), 2.05−1.95

(m, 2H), 1.93−1.80 (m, 8H), 1.67−1.53 (m, 4H); ¹³C NMR (126

MHz, DMSO-d₆) δ 155.64, 149.93, 147.90, 147.31, 120.35, 117.97,

114.42, 111.87, 92.78, 75.20, 59.13, 57.77, 49.88, 33.10, 32.63, 32.06,

25.04; HRMS (ESI) m/z: [M + H]⁺Calcd for C₂₂H₂₉N₆O 393.2397;

Found: 393.2397.

1

piperidine-1-carboxylate. LCMS: m/z = 380.2 [M + H]⁺; H NMR

(400 MHz, DMSO-d₆) δ 9.69 (s, 1H), 8.34 (d, J = 5.3 Hz, 1H), 7.76

(s, 1H), 6.94 (dd, J = 5.2, 1.6 Hz, 1H), 6.02 (s, 1H), 4.73−4.65 (m,

1H), 3.08−2.92 (m, 2H), 2.86−2.77 (m, 1H), 2.70−2.56 (m, 3H),

2.09−1.97 (m, 2H), 1.93−1.82 (m, 4H), 1.81−1.69 (m, 2H), 1.70−

1.61 (m, 2H), 1.51−1.32 (m, 2H).

2-((1-Cyclopentyl-5-(piperidin-4-yl)-1H-pyrazol-3-yl)amino)-

isonicotinonitrile (8). A mixture of 45 (1.20 g, 0.350 mmol), 2-

bromoisonicotinonitrile (175 mg, 0.385 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (16 mg, 0.026 mmol),

tris(dibenzylideneacetone)dipalladium(0) (16 mg, 0.018 mmol) and

sodium tert-butoxide (47 mg, 0.49 mmol) in 1,4-dioxane (12 mL) was

stirred at 80 °C for 16 h. The reaction mixture was ﬁltered and diluted

with water (15 mL). The resulting solution was extracted with ethyl

acetate (2 × 30 mL). The collected organic was dried over anhydrous

sodium sulfate, ﬁltered, and concentrated under reduced pressure. The

residue was dissolved in dichloromethane (15 mL), and a 4 M solution

of hydrogen chloride 1,4-dioxane (0.80 mL, 3.2 mmol) was added at

23 °C. After 16 h, the reaction mixture was concentrated under

reduced pressure, and a portion of the crude product (117 mg, 0.35

mmol) was puriﬁed by preparative reverse-phase HPLC to aﬀord the

title compound as an oﬀ-white solid (54 mg, 46%). LCMS: m/z =

337.2 [M + H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.80 (s, 1H), 8.32

(d, J = 5.2 Hz, 1H), 7.55 (s, 1H), 7.03 (d, J = 5.2 Hz, 1H), 6.15 (s,

1H), 4.81−4.59 (m, 1H), 3.08 (t, J = 11.9, 3.8 Hz, 1H), 2.97 (t, J =

12.9, 2.8 Hz, 2H), 2.05−1.83 (m, 10H), 1.79−1.59 (m, 5H).

2-(1-(3,3-Diﬂuorocyclopentyl)-5-(1-(oxetan-3-yl)piperidin-4-yl)-

1H-pyrazol-3-ylamino)isonicotinonitrile (12). To a solution of 50

(0.40 g, 0.83 mmol) in N,N-dimethylformamide (4 mL) was added 2-

aminoisonicotinonitrile (198 mg, 1.66 mmol), N¹,N²-dimethylcyclo-

hexane-1,2-diamine (24 mg, 0.17 mmol), potassium phosphate (529

mg, 2.49 mmol), and copper iodide (32 mg, 0.17 mmol) and purged

with nitrogen. The resulting solution was heated at 160 °C under

microwave irradiation for 1 h. The mixture was diluted with ethyl

acetate (20 mL) and ﬁltered. The ﬁltrate was extracted with ethyl

acetate (3 × 20 mL). The collected organic was washed with saturated

aqueous sodium chloride solution (20 mL), dried over sodium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by ﬂash

column chromatography (3:1 petroleum ether/ethyl acetate) provided

tert-butyl 4-(3-((4-cyanopyridin-2-yl)amino)-1-(3,3-diﬂuorocyclopen-

tyl)-1H-pyrazol-5-yl)piperidine-1-carboxylate (80 mg). To an ice-

cooled solution of tert-butyl 4-(3-((4-cyanopyridin-2-yl)amino)-1-(3,3-

diﬂuorocyclopentyl)-1H-pyrazol-5-yl)piperidine-1-carboxylate (80 mg,

0.17 mmol) in dichloromethane (10 mL) was added 2,2,2-triﬂuoro-

acetic acid (3 mL). The mixture was stirred at 23 °C for 3 h and

concentrated under reduced pressure. To 2-((1-(3,3-diﬂuorocyclopen-

tyl)-5-(piperidin-4-yl)-1H-pyrazol-3-yl)amino)isonicotinonitrile (65

mg, 0.17 mmol) in methanol (10 mL) was added oxetan-3-one (25

mg, 0.32 mmol) and acetic acid (200 μL), and the resulting mixture

was stirred at 23 °C for 1 h. Sodium cyanoborohydride (32 mg, 0.51

mmol) was added at 23 °C under nitrogen, and the mixture was stirred

at 23 °C for an additional 3 h. The mixture was quenched with water

(15 mL) and extracted with ethyl acetate (3 × 20 mL). The combined

organic was dried over anhydrous sodium sulfate, concentrated under

reduced pressure, and puriﬁed by preparative reverse-phase HPLC to

aﬀord the title compound as a white solid (10.1 mg, 13.9%). LCMS:

m/z = 429.0 [M + H]⁺; ¹H NMR (400 MHz, CD₃OD) δ 8.27 (d, J =

5.2 Hz, 1H), 7.78 (s, 1H), 6.94 (d, J = 4.4 Hz, 1H), 6.05 (s, 1H),

4.92−4.90 (m, 1H), 4.72−4.69 (m, 2H), 4.64−4.61 (m, 2H), 3.64−

3.51 (m, 1H), 2.92−2.87 (m, 2H), 2.81−2.46 (m, 4H), 2.28−2.15 (m,

4H), 2.07−2.00 (m, 2H), 1.94−1.91 (m, 2H), 1.79−1.77 (m, 2H).

2-((5-(1-(Oxetan-3-yl)piperidin-4-yl)-1-(tetrahydrofuran-3-yl)-1H-

pyrazol-3-yl)amino)isonicotinonitrile (13). The title compound (3.4

mg, 3.3% yield) was prepared in a manner analogous to 12 by

2-(5-(1-Acetylpiperidin-4-yl)-1-isopropyl-1H-pyrazol-3-ylamino)-

isonicotinonitrile (9). A mixture of 4 (109 mg, 0.350 mmol), acetic

anhydride (41.0 mg, 0.385 mmol), and triethylamine (0.148 mL, 1.05

mmol) in N,N-dimethylformamide (1.40 mL) was stirred at 40 °C for

16 h. The mixture was concentrated under reduced pressure and

puriﬁed by preparative reverse-phase HPLC to aﬀord the title

compound as a white solid (71 mg, 58%). LCMS: m/z = 353.1 [M

1

+ H]⁺; H NMR (400 MHz, DMSO-d₆) δ 9.83 (s, 1H), 8.31 (d, J =

5.2 Hz, 1H), 7.48 (s, 1H), 7.02 (d, J = 5.1 Hz, 1H), 6.16 (s, 1H), 4.55

(p, J = 6.4 Hz, 1H), 4.51−4.44 (m, 1H), 3.93−3.85 (m, 1H), 3.22−

3.10 (m, 1H), 3.05−2.92 (m, 1H), 2.68−2.57 (m, 1H), 2.03 (s, 3H),

1.84 (s, 2H), 1.60−1.46 (m, 1H), 1.38 (d, J = 6.5 Hz, 6H), 1.35−1.27

(m, 1H).

2-(1-Isopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyrazol-3-

ylamino)isonicotinonitrile (10). A mixture of 4 (109 mg, 0.350 mmol)

and oxetan-3-one (50.0 mg, 0.70 mmol) in dichloromethane (1.75

mL) was stirred at 23 °C for 15 min. Sodium triacetoxyborohydride

(234 mg, 1.05 mmol) was added, and the mixture was stirred at 23 °C

for 1 h. The reaction mixture was partitioned between dichloro-

L

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substituting tert-butyl 4-(3-iodo-1-(tetrahydrofuran-3-yl)-1H-pyrazol-

5-yl)piperidine-1-carboxylate for tert-butyl 4-(1-(3,3-diﬂuorocyclopen-

tyl)-3-iodo-1H-pyrazol-5-yl)piperidine-1-carboxylate. LCMS: m/z =

394.9 [M + H] ⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 4.8 Hz,

1H), 7.59 (s, 1H), 7.01 (s, 1H), 6.92−6.90 (m, 1H), 5.95 (s, 1H),

4.82−4.70 (m, 1H), 4.69−4.62 (m, 4H), 4.23−4.20 (m, 1H), 4.12−

4.09 (m, 1H), 3.94−3.99 (m, 2H), 3.50−3.54 (m, 1H), 2.86−2.89 (m,

2H), 2.60−2.63 (m, 1H), 2.44−2.34 (m, 2H), 1.98−1.79 (m, 6H).

2-((5-(1-(Oxetan-3-yl)piperidin-4-yl)-1-(tetrahydro-2H-pyran-4-

yl)-1H-pyrazol-3-yl)amino)isonicotinonitrile (14). A solution of 52

(450 mg, 0.98 mmol), 2-aminoisonicotinonitrile (233 mg, 1.96 mmol),

N¹,N²-dimethylcyclohexane-1,2-diamine (28 mg, 0.20 mmol), potas-

sium phosphate (623 mg, 2.94 mmol), and copper iodide (38 mg, 0.20

mmol) in N,N-dimethylformamide (4 mL) was purged with nitrogen

and heated at 160 °C under microwave irradiation for 1 h. The mixture

was diluted with ethyl acetate (20 mL) and ﬁltered. The ﬁltrate was

extracted with ethyl acetate (3 × 20 mL). The collected organic was

washed with saturated aqueous sodium chloride solution (20 mL),

dried over sodium sulfate, ﬁltered, and concentrated under reduced

pressure. Puriﬁcation by ﬂash column chromatography (3:1 petroleum

ether/ethyl acetate) provided intermediate tert-butyl 4-(3-((4-

cyanopyridin-2-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-

yl)piperidine-1-carboxylate (60 mg, 0.13 mmol) which was dissolved

in dichloromethane (10 mL) and 2,2,2-triﬂuoroacetic acid (3 mL) at 0

°C. The mixture was warmed to 23 °C for 3 h and concentrated under

reduced pressure. To a solution of crude 2-((5-(piperidin-4-yl)-1-

(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)amino)isonicotinonitrile

(46 mg, 0.13 mmol) in methanol (10 mL) was added oxetan-3-one

(19 mg, 0.26 mmol) and acetic acid (200 μL). The mixture was heated

to 60 °C for 1 h. Sodium cyanoborohydride (25 mg, 0.39 mmol) was

under nitrogen, and the mixture was heated at 60 °C for another 3 h.

The mixture was quenched with water (15 mL) and extracted with

ethyl acetate (3 × 20 mL). The combined organic was dried over

anhydrous sodium sulfate and concentrated under reduced pressure.

Puriﬁcation by preparative reverse-phase HPLC aﬀorded the title

compound as a white solid (15 mg, 28% yield). LC/MS: m/z = 409.15

[M + H]⁺; ¹H NMR (400 MHz, CD₃OD) δ 8.27 (d, J = 5.2 Hz, 1H),

7.69 (s, 1H), 6.94 (d, J = 5.2 Hz, 1H), 6.07 (s, 1H), 4.74−4.70 (m,

2H), 4.65−4.62 (m, 2H), 4.39−4.34 (m, 1H), 4.10−4.06 (m, 2H),

3.65−3.54 (m, 3H), 2.92−2.80 (m, 3H), 2.35−2.25 (m, 2H), 2.08−

2.02 (m, 2H), 1.96−1.93 (m, 2H), 1.83−1.77 (m, 5H).

title compound as a white solid (24 mg, 25% yield). LCMS: m/z =

365.0 [M + H] ⁺; ¹H NMR (400 MHz, CD₃CN) δ 8.28 (d, J = 5.2 Hz,

1H), 8.08 (s, 1H), 7.79 (s, 1H), 6.97 (d, J = 5.2 Hz, 1H), 6.11 (s, 1H),

4.65 (t, J = 6.8 Hz, 2H), 4.47−4.44 (m, 3H), 3.90−3.87 (m, 4H), 3.40

(s, 2H), 2.00−1.93 (m, 3H), 1.91−1.88 (m, 3H), 1.69−1.65 (m, 2H).

2-((1-Cyclopentyl-5-(1-(oxetan-3-yl)pyrrolidin-3-yl)-1H-pyrazol-3-

yl)amino)isonicotinonitrile (17). The title compound (15 mg, 16%)

was prepared in a manner analogous to 14 by substituting tert- tert-

butyl 3-(1-cyclopentyl-3-iodo-1H-pyrazol-5-yl)pyrrolidine-1-carboxy-

late for tert-butyl 4-(3-iodo-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-

1

5-yl)piperidine-1-carboxylate. LCMS: m/z = 378.9 [M + H] ⁺; H

NMR (400 MHz, CD₃OD) δ 8.24 (d, J = 5.2 Hz, 1H), 7.70 (s, 1H),

6.92 (d, J = 5.2 Hz, 1H), 6.08 (s, 1H), 4.77−4.71 (m, 2H), 4.66−4.61

(m, 2H), 3.76−3.73 (m, 1H), 3.57−3.53 (m, 1H), 3.12−3.07 (m, 1H),

2.85−2.79 (m, 1H), 2.68−2.62 (m, 1H), 2.54−2.49 (m, 1H), 2.40−

2.35 (m, 1H), 2.06−1.88 (m, 9H), 1.73−1.69 (m, 2H).

2-((1-Cyclopentyl-5-(tetrahydrofuran-3-yl)-1H-pyrazol-3-yl)-

amino)isonicotinonitrile (18). To a suspension of 54 (160 mg, 0.48

mmol), 2-aminoisonicotinonitrile (75 mg, 0.63 mmol), N¹,N²-

dimethylcyclohexane-1,2-diamine (34 mg, 0.24 mmol), potassium

phosphate (102 mg, 0.48 mmol), and copper iodide (46 mg, 0.24

mmol) in N,N-dimethylformamide (3 mL) was purged with nitrogen

and heated at 160 °C under microwave irradiation for 1 h. The mixture

was diluted with ethyl acetate (20 mL) and ﬁltered. The ﬁltrate was

extracted with ethyl acetate (2 × 20 mL), and the collected organic

was washed with saturated aqueous sodium chloride solution (20 mL),

dried over sodium sulfate, ﬁltered, and concentrated under reduced

pressure. Puriﬁcation by preparative reverse-phase HPLC aﬀorded the

title compound as white solid (11 mg, 7%). LCMS: m/z = 324.0 [M +

1

H]⁺; H NMR (400 MHz, CD₃OD) δ 8.41 (d, J = 6.8 Hz, 1H), 7.67

(s, 1H), 7.28 (d, J = 6.8 Hz, 1H), 6.05 (s, 1H), 4.83−4.80 (m, 1H),

4.13−4.09 (m, 1H), 4.02−3.97 (m, 1H), 3.93−3.87 (m, 1H), 3.77−

3.67 (m, 2H), 2.48−2.40 (m, 1H), 2.16−2.09 (m, 5H), 2.02−1.96 (m,

3H), 1.81−1.72 (m, 2H).

2-((4-Cyclopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)thiazol-2-yl)-

amino)isonicotinonitrile (19). A mixture of 61 (0.10 g, 0.36 mmol), 2-

chloroisonicotinonitrile (0.050 g, 0.36 mmol), potassium phosphate

(0.230 g, 1.08 mmol), and Pd(t-Bu₃P)₂(0.010 mg, 0.020 mmol) in

1,4-dioxane (5 mL) was stirred at 100 °C for 3 h. After cooling to 23

°C, the mixture was extracted with ethyl acetate (2 × 30 mL). The

collected organic was washed with brine (50 mL), dried over sodium

sulfate, ﬁltered, and concentrated under reduced pressure. The residue

was puriﬁed by preparative reverse-phase HPLC to aﬀord the title

compound as white solid (7 mg, 5%). LCMS: m/z = 382.2 [M + H]⁺;

¹H NMR (400 MHz, CD₃OD) δ 8.42 (d, J = 4.0 Hz, 1H), 7.27 (s,

2-(1-(Cyclobutylmethyl)-5-(1-(oxetan-3-yl)piperidin-4-yl)-1H-pyr-

azol-3-ylamino)isonicotinonitrile (15). A solution of 41 (100 mg, 0.3

mmol) and oxetan-3-one (43.0 mg, 0.595 mmol) in dichloromethane

(2.4 mL) was stirred at 23 °C for 15 min. Sodium triacetoxyborohy-

dride (199 mg, 0.892 mmol) was added, and the resulting suspension

was maintained at 23 °C for an additional 1 h. The reaction mixture

was partitioned between dichloromethane (15 mL) and saturated

aqueous ammonium chloride solution (15 mL). The organic was

separated, and the aqueous layer was further extracted with

dichloromethane (2 × 5 mL). The collected organic was dried over

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. Puriﬁcation by preparative reverse-phase HPLC aﬀorded the

title compound as a white solid (10 mg, 8%). LCMS: m/z = 393.3 [M

1H), 7.07−7.06 (m, 1H), 4.76−4.72 (m, 2H), 4.69−4.65 (m, 2H),

3.76−3.69 (m, 1H), 3.31−3.11 (m, 1H), 3.05−3.02 (m, 2H), 2.26−

2.21 (m, 2H), 2.05−2.02 (m, 2H), 1.93−1.78 (m, 4H), 0.95−0.75 (m,

4H).

2-((2-Cyclopentyl-1-(1-(oxetan-3-yl)piperidin-4-yl)-1H-imidazol-

4-yl)amino)isonicotinonitrile (20). The title compound (3.5 mg,

7.9%) was prepared in a manner analogous to 14 by substituting tert-

butyl 4-(2-cyclopentyl-4-iodo-1H-imidazol-1-yl)piperidine-1-carboxy-

late for tert-butyl 4-(3-iodo-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-

5-yl)piperidine-1-carboxylate. LCMS: m/z = 393.0 [M + H]⁺; ¹H

NMR (400 MHz, CD₃OD) δ 8.27 (d, J = 5.2 Hz, 1H), 7.24 (s, 1H),

7.04 (s, 1H), 6.88−6.87 (m, 1H), 4.74−4.70 (m, 2H), 4.64−4.61 (m,

2H), 4.18−4.16 (m, 1H), 3.60−3.56 (m, 1H), 3.27−3.25 (m, 1H),

2.96−2.93 (m, 2H), 2.13−2.00 (m, 9H), 1.87−1.84 (m, 4H), 1.72−

1.69 (m, 2H).

tert-Butyl 4-(2-Cyanoacetyl)piperidine-1-carboxylate (25). To a

stirred solution of 1-tert-butyl 4-methylpiperidine-1,4-dicarboxylate

(21) (50.0 g, 206 mmol) in THF (1 L) was added acetonitrile (53.0

mL, 103 mmol). The reaction was cooled to 0 °C and potassium tert-

butoxide (69.0 g, 616 mmol) was added portion-wise. The resulting

mixture was stirred at 23 °C for 1 h. The reaction mixture was

quenched with saturated ammonium chloride (2 L) and extracted with

ethyl acetate (3 × 500 mL). The collected organic was sequentially

washed with saturated aqueous sodium chloride solution, dried over

sodium sulfate, ﬁltered, and concentrated under reduced pressure to

1

+ H]⁺; H NMR (400 MHz, DMSO-d₆) δ 9.68 (s, 1H), 8.32 (d, J =

5.2 Hz, 1H), 7.64 (s, 1H), 7.03 (d, J = 4.8 Hz, 1H), 6.11 (s, 1H), 4.54

(t, J = 6.5 Hz, 2H), 4.44 (t, J = 6.1 Hz, 2H), 3.98 (d, J = 7.1 Hz, 2H),

3.41 (p, J = 6.5 Hz, 1H), 2.87−2.58 (m, 4H), 2.05−1.72 (m, 10H),

1.64−1.49 (m, 2H).

2-((1-Cyclopentyl-5-(1-(oxetan-3-yl)azetidin-3-yl)-1H-pyrazol-3-

yl)amino)isonicotinonitrile (16). A mixture of 42 (0.080 g, 0.26

mmol) and oxetan-3-one (37 mg, 0.52 mmol) in dichloromethane (3

mL) was stirred at 23 °C for 15 min. Sodium triacetoxyborohydride

(49 mg, 0.78 mmol) was added at 23 °C. After 1 h, the reaction

mixture was partitioned between dichloromethane (15 mL) and

saturated aqueous ammonium chloride solution (15 mL). The organic

was separated, and the aqueous layer was further extracted with

dichloromethane (2 × 5 mL). The collected organic was dried over

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. Puriﬁcation by preparative reverse-phase HPLC aﬀorded the

M

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aﬀord the title compound as a pale yellow oil (40 g, 77% crude yield).

TLC (R_f= 0.5 in 2:1 petroleum ether/ethyl acetate).

over sodium sulfate, and concentrated under reduced pressure to

aﬀord the title compound as a yellow solid (15.8 g, 90% crude yield).

LCMS: m/z = 253.1 [M + H]⁺.

tert-Butyl 3-(2-Cyanoacetyl)azetidine-1-carboxylate (26). To an

ice-cooled solution of 1-tert-butyl 3-methyl azetidine-1,3-dicarboxylate

(22) (10.0 g, 46.5 mmol) and acetonitrile (2.90 g, 69.8 mmol) in

tetrahydrofuran (250 mL) was added potassium tert-butoxide (7.83 g,

69.8 mmol) portion-wise. The resulting mixture was warmed to 23 °C.

After 1 h, the reaction mixture was quenched with saturated aqueous

ammonium chloride solution (1 L), and the resulting solution was

extracted with ethyl acetate (3 × 400 mL). The combined organic was

washed with saturated aqueous sodium chloride solution (2 × 300

mL), dried over sodium sulfate, and concentrated under reduced

pressure to aﬀord the title compound as a light yellow solid (10 g, 96%

crude yield) which was used without further puriﬁcation.

5-(Tetrahydrofuran-3-yl)-1H-pyrazol-3-amine (32). To a stirred

solution of 28 (41.0 g, 0.295 mol) in 2-propanol (400 mL) was added

hydrazine monohydrate (44.2 g, 0.885 mol), and the mixture was

heated at 80 °C for 10 h. The reaction mixture was concentrated under

reduced pressure, and the resulting residue was dissolved in

dichloromethane (500 mL). The organic solution was sequentially

washed with water (3 × 150 mL) and saturated aqueous sodium

chloride solution (200 mL). The collected organic was dried over

sodium sulfate, ﬁltered, and concentrated under reduced pressure to

aﬀord the title compound as a yellow solid (38.5 g, 85% crude yield).

LCMS: m/z = 153.8 [M + H]⁺.

tert-Butyl 3-(2-Cyanoacetyl)pyrrolidine-1-carboxylate (27). To an

ice-cooled solution of 1-tert-butyl 3-methyl pyrrolidine-1,3-dicarbox-

ylate (23) (22.0 g, 96.1 mmol) and acetonitrile (21.0 mL, 480 mmol)

in tetrahydrofuran (440 mL) was added potassium tert-butoxide (32.3

g, 288 mmol) portion-wise. The resulting mixture was warmed to 23

°C and stirred for 1.5 h. The reaction mixture was quenched with

saturated aqueous ammonium chloride solution (1 L), and the

resulting mixture was extracted with ethyl acetate (3 × 500 mL). The

collected organic was washed with saturated aqueous sodium chloride

solution (2 × 500 mL), dried over sodium sulfate, ﬁltered, and

concentrated under reduced pressure to aﬀord the title compound as a

light yellow solid (0.020 kg, 88% crude yield). LCMS: m/z = 239.1 [M

tert-Butyl 4-(3-(1,3-Dioxoisoindolin-2-yl)-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (33). To a stirred solution of 29 (35.0 g,

131 mmol) in dioxane (700 mL) was added phthalic anhydride (19.4

g, 131 mmol). The resulting reaction mixture was heated at 90 °C for

18 h. The reaction mixture was poured into water (2 L) and extracted

with ethyl acetate (3 × 1 L). The collected organic was washed with

saturated aqueous sodium chloride solution, dried over anhydrous

sodium sulfate, ﬁltered, and concentrated under reduced pressure. The

resulting pale yellow viscous oil was triturated with ether to aﬀord the

title compound as a white solid (40 g, 77%). LCMS: m/z = 395.3 [M

− 1]⁻.

tert-Butyl 3-(5-(1,3-Dioxoisoindolin-2-yl)-1H-pyrazol-3-yl)-

azetidine-1-carboxylate (34). A solution of crude 30 (2.0 g, 7.9

mmol) and isobenzofuran-1,3-dione (1.2 g, 7.9 mmol) in dioxane (20

mL) was heated at 110 °C for 2 h. The reaction mixture was

concentrated under reduced pressure and extracted with ethyl acetate

(3 × 80 mL) and water (80 mL). The collected organic was washed

with saturated aqueous sodium chloride solution (80 mL), dried over

sodium sulfate, ﬁltered, and concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (1:2 petroleum ether/

ethyl acetate) aﬀorded the title compound as a white solid (1.5 g,

52%). LCMS: m/z = 369.0 [M + H]⁺.

+

+ H] .

3-Oxo-3-(tetrahydrofuran-3-yl)propanenitrile (28). To an ice-

cooled solution of methyl tetrahydrofuran-3-carboxylate (24) (50.0

g, 0.385 mol) and acetonitrile (47.0 g, 1.15 mol) in tetrahydrofuran

(500 mL) was added potassium tert-butoxide (129 g, 1.15 mol)

portion-wise. The resulting mixture was warmed to 23 °C and stirred

for 1 h. The reaction mixture was quenched with saturated aqueous

ammonium chloride solution (1 L), and the resulting solution was

extracted with ethyl acetate (3 × 400 mL). The collected organic was

washed with saturated aqueous sodium chloride solution (400 mL),

dried over sodium sulfate, ﬁltered, and concentrated under reduced

pressure to aﬀord the title compound (41 g, 77% crude yield).

tert-Butyl 4-(3-Amino-1H-pyrazol-5-yl)piperidine-1-carboxylate

(29). To a solution of 25 (40.0 g, 158 mmol) was added hydrazine

monohydrate (39.6 mL, 792 mmol) in 2-propanol (500 mL) and

heated at 80 °C for 18 h. The reaction mixture was concentrated under

reduced pressure, and the resulting residue was dissolved in

dichloromethane (1 L). The solution was washed sequentially with

water (500 mL) and saturated aqueous sodium chloride solution (500

mL). The collected organic layer was dried over anhydrous sodium

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

title compound as a yellow oil (35 g, 83% crude yield). LCMS: m/z =

267.2 [M + H]⁺; ¹H NMR (400 MHz, DMSO-d₆): δ 11.14 (br s, 1 H),

5.15 (s, 1 H), 4.41 (br s, 2 H), 3.93−3.90 (m, 2 H), 2.76−2.74 (m, 2

H), 2.65−2.55 (m, 1 H), 1.78−1.74 (m, 2 H), 1.39−1.36 (m, 11 H).

tert-Butyl 3-(3-Amino-1H-pyrazol-5-yl)azetidine-1-carboxylate

(30). A solution of 26 (10.0 g, 44.6 mol, crude) and hydrazine

monohydrate (40 mL) in isopropyl alcohol (200 mL) was heated at 80

°C for 16 h. After removal of the solvent under reduced pressure, the

resulting residue was redissolved in dichloromethane (500 mL). The

organic solution was washed sequentially with water (500 mL) and

saturated aqueous sodium chloride solution (500 mL). The collected

organic was dried over sodium sulfate and concentrated under reduced

pressure to aﬀord the title compound as a yellow solid (10 g, 94%

tert-Butyl 4-(3-(1,3-Dioxoisoindolin-2-yl)-1-isopropyl-1H-pyrazol-

5-yl)piperidine-1-carboxylate (35). A sealed tube charged with 33

(10.0 g, 252 mmol), cesium carbonate (24.6 g, 75.8 mmol) and

isopropyl iodide (12.6 mL, 126 mmol) in N,N-dimethylformamide

(200 mL) was heated at 60 °C for 3 h. The reaction mixture was

poured into water (100 mL) and extracted with ethyl acetate (3 × 100

mL). The collected organic was washed with saturated aqueous

sodium chloride solution, dried over anhydrous sodium sulfate, and

concentrated under reduced pressure. Puriﬁcation by ﬂash column

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

compound as a pale white solid (2.2 g, 20%). LCMS: m/z = 439.5

+

[M + H] .

tert-Butyl 4-(1-(Cyclobutylmethyl)-3-(1,3-dioxoisoindolin-2-yl)-

1H-pyrazol-5-yl)piperidine-1-carboxylate (36). A glass tube charged

with 33 (0.500 g, 1.26 mmol), cesium carbonate (823 mg, 2.52 mmol),

and (bromomethyl)cyclobutane (376 mg, 2.52 mmol) in N,N-

dimethylacetamide (10 mL) was sealed and heated at 50 °C for 16

h. The reaction mixture was concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (0% → 100%, heptane/

ethyl acetate) aﬀorded the title compound as an oﬀ-white solid (312

mg, 53%). LCMS: m/z = 465.5 [M + H]⁺.

tert-Butyl 3-(1-Cyclopentyl-3-(1,3-dioxoisoindolin-2-yl)-1H-pyra-

zol-5-yl)azetidine-1-carboxylate (37). A suspension of 34 (1.20 g,

3.26 mmol), bromocyclopentane (970 mg, 6.5 mmol), and cesium

carbonate (2.10 g, 6.52 mmol) in N,N-dimethylformamide (10 mL)

was heated at 100 °C for 3 h under nitrogen. The reaction mixture was

diluted with ethyl acetate (50 mL), and the resulting solid precipitate

was ﬁltered. The ﬁltrate was concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (3:1 petroleum ether/

ethyl acetate) aﬀorded the title compound as a white solid (0.30 g,

21%). LCMS: m/z = 437.1 [M + H]⁺.

1

crude yield). LCMS: m/z = 238.9 [M + H]⁺; H NMR (400 MHz,

DMSO-d₆) δ 11.30 (br s, 1H), 5.28 (s, 1H), 4.68 (br s, 2H), 4.12−

4.08 (m, 2H), 3.90−3.79 (m, 2H), 3.63−3.56 (m, 1H), 1.38 (s, 9H).

tert-Butyl 3-(3-Amino-1H-pyrazol-5-yl)pyrrolidine-1-carboxylate

(31). A solution of 27 (16.5 g, 69.3 mmol, crude) and hydrazine

monohydrate (19.0 mL, 347 mmol) in isopropyl alcohol (400 mL)

was heated to 80 °C for 16 h. After removal of the solvent under

reduced pressure, the residue was redissolved in dichloromethane (500

mL). The organic solution was washed sequentially with water (500

mL) and saturated aqueous sodium chloride solution (500 mL), dried

tert-Butyl 4-(3-Amino-1-isopropyl-1H-pyrazol-5-yl)piperidine-1-

carboxylate (38). To an ice-cooled solution of 35 (28.0 g, 63.9

mmol) in methanol (1 L) was added hydrazine hydrate (9.50 mL, 192

N

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Journal of Medicinal Chemistry

Article

mmol). After stirring at 23 °C for 30 min, the reaction mixture was

concentrated under reduced pressure. The resulting oil was triturated

with ethyl acetate, and the solid was ﬁltered. The ﬁltrate was washed

with water and concentrated under reduced pressure. The resulting oil

was triturated with ether to aﬀord the title compound as a white solid

compound as a yellow oil (1.65 g, 69%). LCMS: m/z = 413.4 [M + H]

.

+

tert-Butyl 4-(3-Amino-1-cyclopentyl-1H-pyrazol-5-yl)piperidine-

1-carboxylate (45). To a solution of potassium hydroxide (0.516 g,

7.82 mmol) in water (7 mL) and ethanol (17 mL) was added

hydroxylamine hydrochloride (1.09 g, 20.0 mmol) and 44 (1.65 g, 4.00

mmol). The mixture was heated at 80 °C. After 16 h, the reaction

mixture was ﬁltered and concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (0 → 100% heptane/

ethyl acetate) aﬀorded the title compound as a white solid (457 mg,

1

(10.5 g, 53%). LCMS: m/z = 309.3 [M + H]⁺; H NMR (400 MHz,

DMSO-d₆): δ 5.15 (s, 1 H), 4.40 (br s, 2 H), 4.31 (m, 1 H), 4.00 (m, 2

H), 2.70−2.89 (m, 3 H), 1.74 (m, 2 H), 1.41 (s, 9 H), 1.32 (m, 2 H),

1.27 (d, 6 H).

tert-Butyl 4-(3-Amino-1-(cyclobutylmethyl)-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (39). To a solution of 36 (312 mg, 0.672

mmol) in methanol (2.7 mL) was added hydrazine hydrate (103 mg,

2.01 mmol) at 23 °C. After 16 h, the reaction mixture was

concentrated under reduced pressure to aﬀord the title compound

as a dark yellow oil (224 mg, 99% crude yield). LCMS: m/z = 335.5

+

34%). LCMS: m/z = 335.3 [M + H] .

tert-Butyl 4-(3-Iodo-1H-pyrazol-5-yl)piperidine-1-carboxylate

(46). To an ice-cooled solution of 29 (35.5 g, 133 mmol) in

acetonitrile/water (5:1, 500 mL) was added p-toluenesulfonic acid

monohydrate (70.3 g, 414 mmol) and sodium nitrite (17.2 g, 250

mmol). After 30 min, sodium iodide (60.0 g, 400 mmol) in water (85

mL) was added at 0 °C. After 30 min, the reaction mixture was

warmed to 23 °C. After 1 h, the reaction mixture was poured into

water (1500 mL) and extracted with ethyl acetate (3 × 1500 mL). The

combined organic layer was washed with saturated aqueous sodium

chloride solution (1000 mL), dried over anhydrous sodium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by ﬂash

column chromatography (25% ethyl acetate in petroleum ether)

+

[M + H] .

tert-Butyl 3-(3-Amino-1-cyclopentyl-1H-pyrazol-5-yl)azetidine-1-

carboxylate (40). A mixture of 37 (0.30 g, 0.69 mmol) in ethanol (10

mL), hydrazine monohydrate (10 mL), and water (10 mL) was heated

at 110 °C for 2 h. The reaction mixture was concentrated under

reduced pressure, and the resulting aqueous solution was extracted

with ethyl acetate (3 × 50 mL). The collected organic was

concentrated under reduced pressure to aﬀord the title compound

as a yellow oil (150 mg, 71% crude yield). LCMS: m/z = 306.9 [M +

H]⁺.

1

aﬀorded the title compound as a yellow solid (11.4 g, 23% yield). H

NMR (400 MHz, CDCl₃): δ 6.21 (s, 1H), 4.14−4.12 (m, 2H), 3.10−

3.02 (m, 1H), 2.90−2.80 (m, 3H), 1.92−1.89 (m, 2H), 1.62−1.52 (m,

2H), 1.46 (s, 9H).

2-((1-(Cyclobutylmethyl)-5-(piperidin-4-yl)-1H-pyrazol-3-yl)-

amino)isonicotinonitrile (41). A suspension of 39 (0.200 g, 0.598

mmol), 2-bromoisonicotinonitrile (109 mg, 0.598 mmol), 4,5-

bis(diphenylphosphino)-9,9-dimethylxanthene (27 mg, 0.045 mmol),

tris(dibenzylideneacetone)dipalladium(0) (28 mg, 0.031 mmol), and

sodium tert-butoxide (83 mg, 0.84 mmol) in 1,4-dioxane (3.2 mL) was

heated at 80 °C for 1 h. The reaction mixture was ﬁltered and diluted

with water (5 mL). The resulting solution was extracted with ethyl

acetate (2 × 3 mL). The collected organic was concentrated under

reduced pressure and puriﬁed by preparative reverse-phase HPLC

(149 mg, 57%). The isolated compound (149 mg, 0.342 mmol) was

dissolved in dichloromethane (2.5 mL) and a 4.0 M solution of 1,4-

dioxane (0.8 mL, 3.2 mmol) at 23 °C. After 3 h, the reaction mixture

was concentrated under reduced pressure to aﬀord the title compound

as a yellow oil (100 mg, 87% crude yield) which was used without

tert-Butyl 3-(3-Iodo-1H-pyrazol-5-yl)pyrrolidine-1-carboxylate

(47). To an ice-cooled solution of 31 (5.0 g, 20 mmol) in

acetonitrile/water (4:1, 75 mL) was added p-toluenesulfonic acid

monohydrate (10.2 g, 60.0 mmol) and sodium nitrite (2.8 g, 40

mmol). After 30 min, sodium iodide (9.0 g, 60 mmol) in water (15

mL) was added. After an additional 30 min, the reaction mixture was

allowed to warm to 23 °C and stirred for 1 h. The reaction mixture

was poured into water (500 mL), and the resulting solution was

extracted with ethyl acetate (3 × 500 mL). The collected organic was

washed with saturated aqueous sodium chloride solution (500 mL),

dried over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation by ﬂash column chromatography (20%

ethyl acetate in petroleum ether) yielded the title compound as a

yellow solid (2.9 g, 40% yield). LCMS: m/z = 364.1 [M + H]⁺.

3-Iodo-5-(tetrahydrofuran-3-yl)-1H-pyrazole (48). To an ice-

cooled solution of 32 (18.0 g, 0.118 mmol) in 5:1 acetonitrile/water

(240 mL) was added p-toluenesulfonic acid monohydrate (30.0 g,

0.176 mmol) and sodium nitrite (12.2 g, 0.176 mmol). After 30 min,

sodium iodide (26.5 g, 0.176 mmol) was slowly added, and the

reaction was warmed to 23 °C for 1 h. The reaction mixture was

diluted with water (600 mL), and the resulting solution was extracted

with ethyl acetate (3 × 300 mL). The collected organic was washed

with saturated aqueous sodium chloride solution (500 mL), dried over

sodium sulfate, ﬁltered, and concentrated under reduced pressure.

Puriﬁcation of the resulting residue by ﬂash column chromatography

(2:1 petroleum ether/ethyl acetate) aﬀorded the title compound as a

yellow solid (11 g, 35%). ¹H NMR (400 MHz, CDCl₃) δ 10.50 (br s,

1H), 5.73 (s, 1H), 3.99−3.92 (m, 2H), 3.86−3.80 (m, 1H), 3.78−3.72

(m, 1H), 3.50−3.38 (m, 1H), 2.40−2.26 (m, 1H), 1.98−1.95 (m, 1H).

tert-Butyl 4-(3-Iodo-1-(3-oxocyclopentyl)-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (49). To a solution of 46 (6.0 g, 16

mmol) in dichloromethane (200 mL) was added cyclopent-2-enone

(3.9 g, 48 mmol) and hafnium chloride (512 mg, 1.60 mmol). The

resulting mixture was stirred at 23 °C for 12 h. The mixture was

ﬁltered, and the ﬁltrate was concentrated under reduced pressure to

aﬀord the title compound as dark yellow oil (7.3 g, 100% crude yield).

+

further puriﬁcation. LCMS: m/z = 337.4 [M + H] .

2-((5-(Azetidin-3-yl)-1-cyclopentyl-1H-pyrazol-3-yl)amino)-

isonicotinonitrile (42). The title compound (0.080 g, 78%) was

prepared in a manner analogous to 3 by substituting tert-butyl 3-(3-

amino-1-cyclopentyl-1H-pyrazol-5-yl)azetidine-1-carboxylate and 2-

bromoisonicotinonitrile for tert-butyl 4-(3-amino-1-isopropyl-1H-pyr-

azol-5-yl)piperidine-1-carboxylate and 2-bromo-4-(triﬂuoromethyl)-

pyridine, respectively. LCMS: m/z = 308.9 [M + H]⁺.

tert-Butyl 4-(3-(2,5-Dimethyl-1H-pyrrol-1-yl)-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (43). To a solution of 29 (1.60 g, 6.01

mmol) in toluene (4 mL) was added 2,5-hexanedione (1.30 g, 11.4

mmol) and magnesium sulfate (0.723 g, 6.01 mmol). The reaction

mixture was heated at 40 °C. After 16 h, the reaction was diluted with

water (5 mL) and ethyl acetate (5 mL). The resulting mixture was

ﬁltered, and the ﬁltrate was concentrated under reduced pressure to

aﬀord a yellow oil (2.01 g, 97% crude yield). LCMS: m/z = 345.3 [M

+ H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 12.66 (s, 1H), 6.08 (s, 1H),

5.72 (s, 2H), 4.11−3.90 (m, 2H), 2.95−2.76 (m, 3H), 2.01 (s, 6H),

1.96−1.86 (m, 2H), 1.58−1.43 (m, 2H), 1.41 (s, 9H).

tert-Butyl 4-(1-Cyclopentyl-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-

pyrazol-5-yl)piperidine-1-carboxylate (44). To a solution of 43 (2.0

g, 5.8 mmol) in N,N-dimethylacetamide (48 mL) was added

iodocyclopentane (2.4 g, 12 mmol) and cesium carbonate (5.9 g, 18

mmol), and the mixture was heated at 50 °C for 16 h. The reaction

mixture was diluted with water (50 mL) and extracted with ethyl

acetate (3 × 50 mL). The collected organic was washed with water (20

mL), saturated aqueous sodium chloride solution (20 mL), and

concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography (0 → 100% heptane/ethyl acetate) aﬀorded the title

+

LCMS: m/z = 359.8 [MH-100] .

tert-Butyl 4-(1-(3,3-Diﬂuorocyclopentyl)-3-iodo-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (50). To a crude mixture of 49 (7.30 g, 15.9

mmol) in dichloromethane (200 mL) was added DAST (15 mL) at 0

°C. The mixture was allowed to warm to 23 °C and stirred for 12 h.

Saturated aqueous sodium bicarbonate solution (60 mL) was added

dropwise, and the resulting mixture was extracted with petroleum

O

DOI: 10.1021/acs.jmedchem.5b01072

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Journal of Medicinal Chemistry

Article

ether (3 × 80 mL). The collected organic was washed with saturated

aqueous sodium chloride solution (80 mL), dried over sodium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by ﬂash

column chromatography (4:1 petroleum ether/ethyl acetate) aﬀorded

the title compound as a white solid (1.5 g, 20% yield). R_f= 0.3 in 5:1

aﬀorded the title compound as a yellow oil (980 mg, 85% yield).

LCMS: m/z = 218.8 [M − 56].

tert-Butyl 4-(2-((tert-Butoxycarbonyl)amino)-4-cyclopropylthia-

zol-5-yl)-5,6-dihydropyridine-1(2H)-carboxylate (58). A suspension

of 57 (0.700 g, 2.55 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (789 mg,

2.55 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (100

mg, 0.200 mmol), tris(dibenzylideneacetone)dipalladium(0) (70 mg,

0.077 mmol), and sodium carbonate (811 mg, 7.65 mmol) in 3:1 1,4-

dioxane/water (20 mL) was stirred at 100 °C for 4 h. After cooling to

23 °C, the mixture was extracted with ethyl acetate (3 × 30 mL). The

collected organic layer was washed with brine (50 mL), dried over

sodium sulfate, ﬁltered, and concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (10:1 petroleum ether/

ethyl acetate) aﬀorded the title compound as brown oil (0.50 g, 47%).

LCMS: m/z = 365.9 [M − 56].

1

petroleum ether/ethyl acetate); H NMR (400 MHz, CDCl₃) δ 6.14

(s, 1H), 4.69−4.65 (m, 1H), 4.23−4.21 (m, 2H), 2.85−2.77 (m, 3H),

2.73−2.67 (m, 1H), 2.55−2.47 (m, 2H), 2.40−2.35 (m, 1H), 2.26−

2.14 (m, 2H), 1.81−1.78 (m, 2H), 1.56−1.53 (m, 2H), 1.47 (s, 9H).

tert-Butyl 4-(3-Iodo-1-(tetrahydrofuran-3-yl)-1H-pyrazol-5-yl)-

piperidine-1-carboxylate (51). To an ice-cooled solution of 46 (2.0

g, 5.3 mmol) in N,N-dimethylformamide (30 mL) was added sodium

hydride (637 mg, 26.5 mmol, 60% in mineral oil). After 15 min,

tetrahydrofuran-3-yl methanesulfonate (2.03 g, 12.2 mmol) was added

dropwise, and the reaction mixture was allowed to warm to 23 °C.

After 1 h, the mixture was heated to 90 °C for 2 h. The reaction

mixture was quenched with methanol (20 mL) at 23 °C, and the

resulting solution was extracted with ethyl acetate (3 × 60 mL). The

combined organic was washed with saturated aqueous sodium chloride

solution (300 mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography (0 → 30% ethyl acetate in petroleum ether) aﬀorded

the title compound as white solid (730 mg, 31% yield). R_f= 0.3 in 2:1

petroleum ether/ethyl acetate.

tert-Butyl 4-(2-((tert-Butoxycarbonyl)amino)-4-cyclopropylthia-

zol-5-yl)piperidine-1-carboxylate (59). A mixture of 58 (0.50 g, 1.2

mmol) and 10% palladium on carbon (500 mg, 0.470 mmol) in

methanol (50 mL) was stirred at 23 °C under hydrogen (55 psi) for

16 h. The reaction mixture was ﬁltered through Celite, and the ﬁltrate

was concentrated under reduced pressure to aﬀord the title compound

as a white solid (490 mg, 98% crude yield). LCMS: m/z = 424.2 [M +

H]⁺.

4-Cyclopropyl-5-(piperidin-4-yl)thiazol-2-amine (60). To an ice-

cooled solution of 59 (0.200 g, 0.474 mmol) in dichloromethane (5

mL) was added 2,2,2-triﬂuoroacetic acid (1.20 mL, 9.48 mmol) under

nitrogen. The reaction mixture was warmed to 23 °C. After 4 h, the

reaction mixture was concentrated under reduced pressure to aﬀord

the title compound as a yellow solid (118 mg, 100% crude yield).

LCMS: m/z = 223.9 [M + H]⁺.

4-Cyclopropyl-5-(1-(oxetan-3-yl)piperidin-4-yl)thiazol-2-amine

(61). A mixture of 60 (0.100 g, 0.448 mmol) and oxetan-3-one (64.0

mg, 0.897 mmol) in methanol (5.0 mL) was stirred at 23 °C for 1 h.

Sodium cyanoborohydride (83.0 mg, 1.34 mmol) was added, and the

reaction was maintained at 23 °C for an additional 3 h. Water (20 mL)

was added, and the mixture was extracted with dichloromethane (2 ×

20 mL). The collected organic was washed with brine (50 mL), dried

over sodium sulfate, ﬁltered, and concentrated under reduced pressure

to aﬀord the title compound as a yellow oil (78 mg, 62% crude yield),

which was used without further puriﬁcation. LCMS: m/z = 280.0 [M +

H]⁺.

2-Cyclopentyl-4,5-diiodo-1H-imidazole (63). To a solution of 2-

cyclopentyl-1H-imidazole (62) (2.00 g, 14.6 mmol) in chloroform (30

mL) was added iodine (3.73 g, 29.2 mmol) and followed by a 4 M

solution of sodium hydroxide (30 mL). The mixture was heated at 50

°C for 6 h. After cooling to 23 °C, the reaction was quenched with

saturated sodium sulﬁte solution (50 mL). The organic layer was

concentrated under reduced pressure to aﬀord the title compound (2.0

g, 35% crude yield). LCMS: m/z = 389.1 [M + H]⁺.

2-Cyclopentyl-4-iodo-1H-imidazole (64). To a solution of 63 (2.0

g, 5.2 mmol) in 30% ethanol (30 mL) was added sodium sulﬁte (1.30

g, 10.4 mmol), and the mixture was reﬂuxed for 10 h. The reaction

mixture was concentrated under reduced pressure, and the residue was

puriﬁed by ﬂash column chromatography (4:1 petroleum ether/ethyl

acetate) to aﬀord the title compound as a white solid (1.0 g, 74%).

LCMS: m/z = 263.8 [M + H]⁺.

tert-Butyl 4-(3-Iodo-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-

yl)piperidine-1-carboxylate (52). To an ice-cooled solution of 46

(1.00 g, 2.65 mmol) in N,N-dimethylformamide (20 mL) was added

sodium hydride (127 mg, 5.30 mmol, 60% in mineral oil). After 30

min, tetrahydro-2H-pyran-4-yl methanesulfonate (954 mg, 5.30

mmol) was added to the reaction, and the mixture was purged with

nitrogen and warmed to 60 °C. After 4 h, the reaction was cooled to

23 °C and quenched with methanol (20 mL). The resulting solution

was concentrated under reduced pressure. Puriﬁcation by ﬂash column

chromatography (3:1 petroleum ether/ethyl acetate) aﬀorded the title

compound as a white solid (450 mg, 38% yield). LCMS: m/z = 461.9

[M + H]⁺.

tert-Butyl 3-(1-Cyclopentyl-3-iodo-1H-pyrazol-5-yl)pyrrolidine-1-

carboxylate (53). A suspension of 47 (1.00 g, 2.75 mmol),

bromocyclopentane (820 mg, 5.5 mmol), and cesium carbonate

(1.79 g, 5.51 mmol) in N,N-dimethylformamide (5 mL) was heated at

50 °C for 2 h under nitrogen. The reaction mixture was diluted with

ethyl acetate (20 mL), and the resulting precipitate was ﬁltered. The

ﬁltrate was concentrated under reduced pressure. Puriﬁcation by ﬂash

column chromatography (3:1 petroleum ether/ethyl acetate) aﬀorded

the title compound as a white solid (0.30 g, 25%). LCMS: m/z = 432.1

+

[M + H] .

1-Cyclopentyl-3-iodo-5-(tetrahydrofuran-3-yl)-1H-pyrazole (54).

A suspension of 48 (0.50 g, 1.9 mmol), bromocyclopentane (840

mg, 5.68 mmol), and potassium carbonate (780 mg, 5.68 mmol) in

N,N-dimethylformamide (10 mL) was heated at 80 °C for 3 h under

nitrogen. The reaction mixture was diluted with ethyl acetate (20 mL),

and the resulting precipitate was ﬁltered. The ﬁltrate was concentrated

under reduced pressure. Puriﬁcation by ﬂash column chromatography

(3:1 petroleum ether/ethyl acetate) aﬀorded the title compound as a

white solid (0.20 mg, 32%). LCMS: m/z = 332.8 [M + H]⁺.

tert-Butyl (4-Cyclopropylthiazol-2-yl)carbamate (56). A neat

mixture of 4-cyclopropylthiazol-2-amine (55) (2.90 g, 20.7 mmol)

and di-tert-butyl dicarbonate (45.0 g, 20.7 mmol) was heated at 80 °C

for 5 h. Puriﬁcation by ﬂash column chromatography (4:1 petroleum

ether/ethyl acetate) aﬀorded the title compound as a white solid (3.69

g, 74% yield). LCMS: m/z = 240.8 [M + H]⁺.

tert-Butyl (5-Chloro-4-cyclopropylthiazol-2-yl)carbamate (57).

To an ice-cooled solution of 56 (1.00 g, 4.17 mmol) in dichloro-

methane (20 mL) was added 1-chloropyrrolidine-2,5-dione (563 mg,

4.17 mmol) portion-wise. After 2 h at 0 °C, the mixture was diluted

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

with water (3 × 50 mL). The collected organic was dried over sodium

sulfate, ﬁltered, and concentrated under reduced pressure. Puriﬁcation

by ﬂash column chromatography (10:1 petroleum ether/ethyl acetate)

tert-Butyl 4-(2-Cyclopentyl-4-iodo-1H-imidazol-1-yl)piperidine-1-

carboxylate (65). To an ice-cooled solution of 64 (1.0 g, 3.8 mmol) in

N,N-dimethylformamide (20 mL) was added sodium hydride (304

mg, 7.60 mmol, 60% in mineral oil). The reaction mixture was warmed

to 23 °C for 30 min before the addition of tert-butyl 4-

((methylsulfonyl)oxy)piperidine-1-carboxylate (3.20 g, 11.4 mmol).

The reaction was purged with nitrogen and heated at 100 °C for 8 h.

After cooling to 23 °C, methanol was added to the reaction, and the

resulting solution was concentrated under reduced pressure.

Puriﬁcation by preparative reverse-phase HPLC aﬀorded the title

compound as a white solid (130 mg, 8%). LCMS: m/z = 446.1 [M +

H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 6.92 (s, 1H), 4.40−4.20 (m, 2H),

P

DOI: 10.1021/acs.jmedchem.5b01072

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Article

4.15−4.00 (m, 1H), 3.05−2.92 (m, 1H), 2.90−2.70 (m, 2H), 1.99−

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1.63 (m, 12H), 1.48 (s, 9H).

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01072.

Protein expression, puriﬁcation, and crystallographic

methods; structure determination and reﬁnement

methods; crystal structure data collection and reﬁnement

statistics and computational methods for lowest energy

conformation analysis (PDF)

(CSV)

Accession Codes

The PDB codes are as follows: 5CEN for Apo DLK kinase

domain, 5CEO for 2 complexed with DLK, 5CEP for 3

complexed with DLK, and 5CEQ for 11 complexed with DLK.

AUTHOR INFORMATION

Corresponding Authors

*S.P.: phone, 650-225-4272; e-mail, patel.snahel@gene.com.

*S.F.H.: phone, 650-467-5187; e-mail, harris.seth@gene.com.

■

Present Addresses

^○College of Pharmacy, Chungnam National University, 99

Daehak-ro, Yuseong-gu, Daejeon 305−764, Republic of Korea

(South)

^◆Well Institute for Cell & Molecular Biology, Cornell

University, Ithaca, New York, United States

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the following individuals for their contributions:

Mengling Wong, Chris Hamman, and Michael Hayes for

compound puriﬁcation; Genentech protein expression group

for the supply of protein; 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.

(12) Ferraris, D.; Yang, Z.; Welsbie, D. Dual leucine zipper kinase as

a therapeutic target for neurodegenerative conditions. Future Med.

Chem. 2013, 5, 1923−34.

(13) Patel, S.; Cohen, F.; Dean, B. J.; De La Torre, K.; Deshmukh,

G.; Estrada, A. A.; Ghosh, A. S.; Gibbons, P.; Gustafson, A.; Huestis,

M. P.; Le Pichon, C. E.; Lin, H.; Liu, W.; Liu, X.; Liu, Y.; Ly, C. Q.;

Lyssikatos, J. P.; Ma, C.; Scearce-Levie, K.; Shin, Y. G.; Solanoy, H.;

Stark, K. L.; Wang, J.; Wang, B.; Zhao, X.; Lewcock, J. W.; Siu, M.

Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors

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ABBREVIATIONS

■

clogP, calculated logarithm of partition coeﬃcient; CNS MPO,

central nervous system multiparameter optimization; DMEM,

Dulbecco’s modiﬁed eagle medium; DLK, dual leucine zippe

kinase; DRG, dorsal root ganglion; DAST, diethylaminosulfur

triﬂuoride; ELISA, enzyme-linked immunosorbent assay; JNK,

c-Jun N-terminal kinase; LipE, lipophilic ligand eﬃciency;

MAP3K12, mitogen-activated protein kinase kinase kinase 12;

MCT, methylcellulose Tween 80; MKK, mitogen-activated

protein kinase kinase; MLK, mixed-lineage kinase; MDR1,

multidrug resistance protein 1; MDCK, Madin−Darby canine

kidney; MSD, mesoscale discovery detection; NGF, nerve

growth factor; P-gp, P-glycoprotein

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protein kinases: structural basis for regulation. Cell 1996, 85, 149−158.

(18) Huse, M.; Kuriyan, J. The conformational plasticity of protein

kinases. Cell 2002, 109, 275−282.

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Scaffold-Hopping and Structure-Based Discovery of Potent, Selective, And Brain Penetrant N-(1H-Pyrazol-3-yl)pyridin-2-amine Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12)

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