Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin- 3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): A highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator

Article

pubs.acs.org/jmc

Discovery and Preclinical Characterization of 1‑Methyl‑3-

(4‑methylpyridin‑3‑yl)‑6‑(pyridin‑2‑ylmethoxy)‑1H‑pyrazolo-

[3,4‑b]pyrazine (PF470): A Highly Potent, Selective, and Eﬃcacious

Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric

Modulator

Lei Zhang,*^,†Gayatri Balan,^∥Gabriela Barreiro,^†Brian P. Boscoe,^∥Lois K. Chenard,^∥Julie Cianfrogna,^#

Michelle M. Claﬀey,^∥Laigao Chen,^○Karen J. Coﬀman,^∥Susan E. Drozda,^∥Joshua R. Dunetz,^⊥

Kari R. Fonseca,^‡Paul Galatsis,^†Sarah Grimwood,^§John T. Lazzaro,^⊥Jessica Y. Mancuso,^∇

Emily L. Miller,^#Matthew R. Reese,^∥Bruce N. Rogers,^†Isao Sakurada,^∥Marc Skaddan,^○

Deborah L. Smith,^§Antonia F. Stepan,^†Patrick Trapa,^‡Jamison B. Tuttle,^†Patrick R. Verhoest,^†

Daniel P. Walker,^∥Ann S. Wright,^∥Margaret M. Zaleska,^§Kenneth Zasadny,^○and Christopher L. Shaﬀer^‡

^†Neuroscience Medicinal Chemistry, ^‡Neuroscience Pharmacokinetics, Dynamics and Metabolism, and ^§Neuroscience Research Unit,

Pﬁzer Inc., Cambridge, Massachusetts 02139, United States

⊥

#

∇

^∥Neuroscience Medicinal Chemistry, Pharmaceutical Sciences, Pharmacokinetics, Dynamics and Metabolism, Research Statistics,

and ^○Bioimaging Center, Pﬁzer Inc., Groton, Connecticut 06340, United States

S

* Supporting Information

ABSTRACT: A novel series of pyrazolopyrazines is herein disclosed as

mGluR5 negative allosteric modulators (NAMs). Starting from a high-

throughput screen (HTS) hit (1), a systematic structure−activity relationship

(SAR) study was conducted with a speciﬁc focus on balancing pharmacological

potency with physicochemical and pharmacokinetic (PK) properties. This

eﬀort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-

ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent,

selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated

robust eﬃcacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-

rendered Parkinsonian nonhuman primate model of ^L-DOPA-induced

dyskinesia (PD-LID). However, the progression of 14 to the clinic was

terminated because of a potentially mechanism-mediated ﬁnding consistent

with a delayed-type immune-mediated type IV hypersensitivity in a 90-day

NHP regulatory toxicology study.

suggesting their potential for treating anxiety, treatment-

resistant depression, addiction, Fragile X Syndrome, and ^L-

DOPA-induced dyskinesia (LID) in Parkinson’s disease (PD)

patients.²Our work speciﬁcally entailed identifying a potent

and selective mGluR5 NAM to modulate excessive glutamate

release in the basal ganglia to ameliorate PD-LID.³Several

mGluR5 NAMs, including MPEP,⁴MTEP,⁵AFQ056,⁶and

ADX48621⁷(Figure 1), attenuate LID in 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian

nonhuman primates (NHP).⁸For AFQ056 and ADX48621,

such ﬁndings were also observed in PD-LID patients, providing

a proof-of-concept for mGluR5 NAMs in this indication.⁹

Like MPEP and MTEP, mGluR5 NAM clinical candidates¹⁰

(e.g., AFQ056, RG7090,¹¹and ADX48621) (Figure 1) contain

INTRODUCTION

■

Glutamate, the major excitatory neurotransmitter in the brain,

exerts its neuromodulatory actions through both ionotropic

(e.g., N-methyl-^D-aspartic acid) and metabotropic glutamate

receptors (mGluR). The eight mGluR subtypes (mGluR1−8)

are classiﬁed into three major groups based on their structure,

preferred signal-transduction mechanism, and pharmacology.

Group I includes postsynaptically located mGluR1 and

mGluR5, which couple to the activation of phospholipase C,

leading to the elevation of intracellular calcium and activation of

protein kinase C that classically results in an increase in

synaptic transmission. Both group II (mGluR2−3) and group

III (mGluR4 and mGluR6−8) receptors are located presynap-

tically and are coupled to the inhibition of adenylate cyclase

activity, typically reducing neurotransmitter release.¹Neuro-

therapeutic interest in mGluR5 negative allosteric modulators

(NAMs) stems from both preclinical and clinical evidence

Received: October 18, 2013

Published: January 6, 2014

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Figure 1. Representative mGluR5 NAMs and alkyne-lacking HTS hit 1.

an alkyne moiety hypothetically susceptible to metabolic

activation that could ultimately cause unacceptable toxicities.

This is particularly important for drugs that have daily clinical

doses >10 mg.¹²Prior to clinical trials, our group evaluated 4-

diﬂuoromethoxy-3-(pyridine-2-ylethynyl)phenyl)-5H-pyrrolo-

[3,4-b]pyridine-6(7H)-yl methanone (GRN-529),¹³an alkyne-

containing mGluR5 NAM developed by Wyeth researchers, in

8-week rat and NHP regulatory toxicology studies. Although it

was safe and well-tolerated in rats (therapeutic index (TI) of 55

at the highest tested dose),¹⁴biliary epithelial hyperplasia was

observed in NHPs at all doses (TI < 4), with increasing severity

at increasing doses (Pﬁzer, unpublished data). Metabolite

proﬁling of NHP hepatic tissue, bile, and plasma samples

revealed extensive glutathione conjugation at the alkyne moiety,

strongly implicating a structural link to the observed

hepatotoxicity. Accordingly, we initiated an eﬀort to identify

novel chemotypes lacking an alkyne motif. Described herein is

the discovery and optimization of a novel class of

pyrazolopyrazines derived from a HTS hit (1), with a speciﬁc

focus on balancing potency with physicochemical and

pharmacokinetic properties guided by central nervous system

multiparameter optimization (CNS MPO),¹⁵ligand eﬃciency

(LE),¹⁶and lipophilicity eﬃciency (LipE).¹⁷This eﬀort

ultimately led to the identiﬁcation of a highly potent, selective,

and orally bioavailable mGluR5 NAM, 1-methyl-3-(4-methyl-

pyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]-

pyrazine (PF470, 14), which demonstrated robust eﬃcacy in a

MPTP-rendered Parkinsonian NHP model of PD-LID.

series. Compound 1 demonstrated good mGluR5 binding (K_i

of 42 nM) and functional inhibitory activity (IC₅₀of 23 nM)

with high functional selectivity over mGluR1 (1543×). Further

examination of historical HTS data revealed that 1 was inactive

in more than 50 other HTS targeting diverse pharmacologies,

suggesting its favorable broad-spectrum selectivity. Additionally,

1 possessed favorable physicochemical properties with a high

CNS MPO (4.83), reasonable LE (0.39) and LipE (3.4), and

many desirable CNS druglike properties,¹⁸including moderate

human liver microsomes-derived intrinsic clearance (HLM

CL_int= 28 mL/min/kg), high passive transcellular permeability

(RRCK P_app,A→B= 19.8 × 10⁻⁶cm/s),¹⁹and no human P-

glycoprotein (P-gp) liability (MDR1 BA/AB = 1.26).²⁰

Importantly, the modular nature of its chemical structure

allowed rapid SAR determination using parallel chemistry. The

overall promising properties of 1, coupled with its chemical

tractability, made it an ideal lead for our chemistry eﬀorts.

Initial SAR studies maintained the pyrazolopyridine core

while exploring variations on both the head amide group

(NR₁R₂) and the ether side chain (R₃) via parallel chemistry

(Table 1). A two-point variation library with over 250

analogues was prepared, which allowed rapid insights into the

series’ general SAR trends. The proﬁles of representative

examples (analogues 2−7) are shown in Table 1. Regarding the

amide SAR, primary amines, such as ethylamine (2), and

amines bearing polar groups (6 and 7) generally led to weaker

potencies. Simple alkyl or cyclic secondary amines, such as

diethyl amine (3 and 4) and 2-methyl pyrrolidine (5), oﬀered

the best potencies and LE, suggesting an interaction of the

amide headgroup with a lipophilic binding pocket. For the tail-

group SAR, the original HTS side chain, 6-methyl-pyridin-2-yl

methyl ether (2 and 3), and the corresponding unsubstituted

pyridin-2-yl methyl ether (4−7) were found to be the most

preferred, oﬀering comparable potency (3 vs 4). However, the

less lipophilic pyridine-2-yl methyl ether provided higher LE,

LipE, and better HLM stability (<8 vs 42.4 mL/min/kg for 4

RESULTS AND DISCUSSION

■

Our eﬀorts to identify novel alkyne-lacking mGluR5 NAMs

began with a HTS of the internal Pﬁzer compound ﬁle, which

yielded a rich collection of chemical matter with 15 structurally

distinct chemotypes. Upon further triaging using mGluR5

potency and CNS MPO, a novel pyrazolopyridine amide series,

exempliﬁed by 1 (Figure 1), emerged as the most promising

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Table 1. mGluR5 Binding (K_i), mGluR5 and mGluR1 Functional Activity (IC₅₀), and Human and Rat Liver Microsomes-

Derived Intrinsic Clearance (CL_int) of Analogues 2−8

a

K_i(geometric mean, n ≥ 3 measurements unless speciﬁed) in HEK-293FT cells expressing human mGluR5 using [³H]MPEPy ([³H]3-methoxy-5-

b

pyridin-2-ylethynylpyridine). IC₅₀(geometric mean, n ≥ 3 unless speciﬁed) in HEK-293 cells expressing rat mGluR5 using ﬂuorimetric imaging

c

d

plate reading (FLIPR). Ligand eﬃciency (LE) and lipophilicity eﬃciency (LipE) calculated using mGluR5 K_i. IC₅₀(geometric mean, n ≥ 3 unless

e

f

speciﬁed) in HEK-293 cells expressing human mGluR1 using FLIPR. Human liver microsomes-derived intrinsic clearance. Rat liver microsomes-

g

derived intrinsic clearance. ND, not determined.

and 3, respectively) and therefore was extensively retained in

subsequent SAR studies. A signiﬁcant species diﬀerence in

microsomal stability was noted for these pyrazolopyridine

analogues; although low-to-moderate HLM CL_intcould be

achieved within the series, rat liver microsomes (RLM)-derived

CL_intwas universally high (>100 mL/min/kg).

Compound 5, an early lead from the initial library, was

further proﬁled in additional in vitro and in vivo PK assays. As

shown in Table 1, 5 maintained the mGluR1 selectivity of 1

while signiﬁcantly improving LE and LipE and lowering HLM

CL_int, but its RLM CL_intremained high (347 mL/min/kg).

Similar to 1, 5 also had high passive permeability (RRCK

P_app,A→B= 21 × 10⁻⁶cm/s) and no P-gp liability (MDR1 BA/

AB = 1.18), which resulted in a rat unbound brain compound

concentration-to-unbound plasma compound concentration

ratio (C_b,u:C_p,u) of 1.01 (Table 2). However, upon further PK

proﬁling, 5 was found to be labile in rat plasma (t_1/2of 5 h)

because of amide hydrolysis, but this instability was not

observed in human plasma. This rat plasma instability together

with its high RLM CL_intresulted in essentially no rat oral

bioavailability (F, 0.4%), which precluded 5 and related

compounds from advancing further to rat safety studies.

To address the poor rat F of the pyrazolopyridine amide

series, we took a two-pronged approach: (A) lower lipophilicity

to improve RLM stability and (B) replace the amide with

appropriate bioisosteres to improve rat plasma stability. For

strategy A, considering that the polarity was generally not

tolerated in the amide side chains, more polar central cores

were incorporated to reduce lipophilicity, which led to the

identiﬁcation of an alternative pyrazolopyrazine core that

resolved the oral bioavailability issue. For example, pyrazolo-

pyrazine 8 (Table 1) had signiﬁcantly improved RLM CL_int

(29.7 mL/min/kg) compared to its corresponding pyrazolo-

pyridine analogue, 4 (978 mL/min/kg), while maintaining

comparable LE and LipE despite a drop in mGluR5 potencies.

Similar to 5, 8 had very high passive permeability, no

susceptibility to P-gp-mediated eﬄux, and a rat C_b,u:C_p,uof

0.51 (Table 2). Serendipitously, no detectable hydrolysis of 8

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analogues (13 and 16) were slightly less active, and more

signiﬁcant potency losses were observed with other 5- and 6-

membered heteroaryls such as methyl pyridazine (18) and

pyrazole (19). As for the heteroaryl substitutions, small alkyls

(13 and 14), haloalkyls (15), haloalkoxys (16), and halogens

(17) oﬀered the best balance of properties, as evidenced by

their mGluR5 potencies and high LE and LipE scores. Bulkier

substituents typically did not provide any beneﬁt in potency but

rather showed deteriorated overall properties. Further inves-

tigation around the N-alkyl substitution at the central pyrazole

ring revealed that methyl was optimal. Larger side chains, such

as ethyl (20) or cyclopropyl (21), led to lower LE and LipE

and elevated HLM CL_int.

Table 2. Select in Vitro and Rat in Vivo Pharmacokinetic

Properties of 5 and 8

From these SAR studies, 1-methyl-3-(4-methylpyridin-3-yl)-

6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (14) was

selected for expanded proﬁling (Figure 2). Compared to the

original HTS hit 1, 14 demonstrated signiﬁcant multifaceted

improvements: it had an almost 50-fold increase in mGluR5 K_i

(0.9 vs 42 nM), it further increased mGluR1 functional

selectivity to >12000-fold, and it showed no apparent

functional agonism (orthosteric or allosteric) or antagonism

activities at any other mGluR up to 50 μM. Broader selectivity

screening with 14 revealed no signiﬁcant activities in kinase

panels (<50% inhibition at 10 uM),²²a proprietary

phosphodiesterase (PDE) panel (IC₅₀> 10 μM),²³and a

broad spectrum BioPrint panel²⁴in which the adenosine A_2A

receptor had the highest aﬃnity (K_i= 6.1 μM). Importantly, its

high potency and selectivity were achieved without compromis-

ing overall physicochemical properties: 14 has a highest

possible CNS MPO score of 6 and is in compliance with the

rule of ﬁve²⁵and the 3/75 analysis (ClogP = 1.5, TPSA= 79

Å²).²⁶A good balance of potency and physicochemical

properties is reﬂected by its high LE and LipE (0.49 and 6.3,

respectively). These physicochemical properties translated into

favorable in vitro PK properties (Table 3), including low-to-

moderate HLM CL_int(23.7 mL/min/kg), high passive

permeability (21 × 10⁻⁶cm/s), and no P-gp liability (MDR1

BA/AB= 1.11).

Intrigued by its exquisite mGluR5 potency and distinct

structure from other known alkyne-containing mGluR5 NAMs,

we were keen to understand how 14 is bound to the mGlu5

receptor. Although an X-ray crystal structure is unavailable, we

were able to gain a preliminary understanding of its binding

mode utilizing an mGluR5 homology model reﬁned by

mutagenesis experiments.²⁷Figure 3 shows a superimposition

of 14 (green) and MPEP (magenta) docked to this mGluR5

homology model that depicts interactions with key residues. In

general, 14 and MPEP appear to occupy the same binding

pocket within mGluR5 but show clear distinctions in their

binding modes. MPEP forms a hydrogen-bond interaction with

residue Y791, and the benzylic ring is sandwiched between

residues W784 and F787. The pyridine ring of MPEP also

makes an edge-to-face π−π interaction with residue Y791.

Analogous to MPEP, 14 makes a similar hydrogen bond with

Y791. However, it also forms a hydrogen-bond interaction with

W784 via the central core. This proposed binding mode

provides a plausible explanation for the essential role of ortho-

methyl substitution for potency. As shown in Figure 3, the

ortho-methyl group torques the pyridyl headgroup orthogonal

to the center core and positions it optimally for a hydrogen

bond with Y791 while nestling itself in a hydrophobic pocket

made by residues F787, L652, and V811 (the last two residues

are not shown for clarity).

a

RRCK cells with low transporter activity were isolated from Mardin-

Darby canine kidney cells and were used to estimate passive

permeability. Ratio from the MS-based quantiﬁcation of P_app,A→B

and P_app,B→Aof the test compound (1 μM) across contiguous

monolayers from MDR1-transfected MDCK cells. Unbound fraction

of compound in rat brain homogenate. Unbound fraction of

compound in rat plasma. AUC_0−Tlast-derived ratio of unbound brain

compound concentration-to-unbound plasma compound concentra-

tion in rats (1 mg/kg, sc). The mean half-life (t_1/2) of the test

b

c

d

e

f

compound (1 μM) following incubation in rat plasma at 37 °C (n =

3). Bioavailability was calculated from the mean observed AUC_0−∞

following single doses of 1 mg/kg iv and 5 mg/kg po (n = 3/dose).

g

was observed in rat plasma (t_1/2> 24 h). Most importantly, the

combination of lower RLM CL_intand rat plasma stability of 8

resulted in much improved rat F (35%).

Concurrent with strategy A, we pursued suitable bioisostere

replacements of the metabolically labile amide to address the

plasma instability of the pyrazolopyridine amides. As outlined

in Table 3, the amide of 5 could be replaced by a phenyl group

without signiﬁcant loss of activity (9). Not surprisingly, the

overall properties of 9, particularly around clearance and passive

permeability, were suboptimal because of its increased

lipophilicity. Nevertheless, it served as a signiﬁcant SAR

inﬂection point, demonstrating that aryls or heteroaryls were

viable amide bioisosteres. Modulating physicochemical proper-

ties by incorporating pyridyl to the headgroup (10 and 11)

considerably improved LipE and increased passive permeability,

but did not lower HLM CL_int. Prompted by the learnings from

strategy A, we incorporated the pyrazolopyrazine core into this

structural series (analogues 12−21). Data for the initial

analogue (12) were mixed. On a positive note, we were

pleasantly surprised to see that this core switch, unlike the

observation from the amide series, did not result in any loss of

mGluR5 activity (12 vs 11). However, HLM CL_intremained

high (94 vs 156 mL/min/kg for 12 and 11, respectively).

Notably, introduction of an ortho-methyl group caused a 5-fold

decrease in HLM CL_int(13 and 14 vs 12) while maintaining

comparable mGluR5 potency and LipE (13 vs 10 and 14 vs

12). We reasoned that this beneﬁt on metabolism might arise

from the conformational changes implicated by the ortho-

substituents, which forced the terminal heteroaryls out of the

plane to minimize the unfavorable steric interaction.²¹

Encouraged by this result, we expanded SAR to various

heteroaryls bearing ortho-substituents. Although many hetero-

aryls were tolerated, 3-pyridyl, as exempliﬁed by 14, 15, and 17,

was optimal for potency. The corresponding 2-pyridyl

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Table 3. SAR of Aryls and Heteroaryls as Amide Replacements

a

K_i(geometric mean, n ≥ 3 unless speciﬁed) in HEK-293FT cells expressing human mGluR5 using [³H]MPEPy ([³H]3-methoxy-5-pyridin-2-

b

ylethynylpyridine). IC₅₀(geometric mean, n ≥ 3 unless speciﬁed) in HEK-293 cells expressing rat mGluR5 using ﬂuorimetric imaging plate reading

(FLIPR). Ligand eﬃciency (LE) and lipophilicity eﬃciency (LipE) calculated using mGluR5 K_i. Human liver microsomes-derived intrinsic

clearance. RRCK cells with low transporter activity were isolated from Mardin-Darby canine kidney cells and were used to estimate passive

permeability. Ratio from the MS-based quantiﬁcation of P_app,A→Band P_app,B→Aof the test compound (1 μM) across contiguous monolayers from

c

d

e

f

MDR1-transfected MDCK cells.

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Figure 2. Potency, selectivity, and physicochemical properties of 14.

Table 4. Rat and Monkey PK Properties of 14

property

rat

monkey

a

b

i

f_u,b

f_u,p

0.091

0.097

2.0

ND

0.064

ND

18

c

C

_b,u:C_p,u

d

CL_b(mL/min/kg)

52

e

V_ss(L/kg)

1.3

0.69

1.4

f

t_1/2(h)

0.31

g

h

F (%)

28

67

a

b

Unbound fraction of compound in brain homogenate. Unbound

c

fraction of compound in plasma. AUC₀₋₄-derived ratio of unbound

brain compound concentration-to-unbound plasma concentration

determined from rat neuropharmacokinetics study (1 mg/kg, sc; n =

d

2/time point). Observed blood clearance (CL_b) following a single iv

e

bolus (1 mg/kg, n = 2/species). Steady-state volume of distribution.

f

g

Half-life. Bioavailability was calculated from the mean observed

AUC_0−∞following single doses of 1 mg/kg iv (n = 2/dose) and 5 mg/

h

kg po (n = 3/dose). Bioavailability was calculated from the mean

observed AUC_0−∞following single doses of 1 mg/kg iv and 30 mg/kg

i

po (n = 2/dose); ND, not determined.

Figure 3. Superimposition of 14 (green) and MPEP (magenta)

docked to the mGluR5 homology model depicting interactions with

key residues. On the basis of the presented putative binding mode

obtained by docking, 14 makes hydrogen bonds to residues W784 and

Y791, and the pyridine ortho-methyl resides in a hydrophobic pocket

made by residues F787, L652, and V811 (not shown). MPEP shows a

hydrogen bond with residue Y791, and the benzyl ring is sandwiched

between residues W784 and F787. The MPEP pyridine also makes an

edge-to-face π−π interaction with residue Y791.

iv infusion regimens of 14 (displacement scans) to maintain

pseudo-steady-state C_p,uover the 120 min scanning period

targeting mGlu5 RO of ∼30, 50, 80, and 90%. An example of a

NHP PET displacement image following a dosing regimen

targeting 80% RO is shown in Figure 4C.³⁰Collectively, in the

NHP PET studies, 14 had a C_p,u-based IC₅₀of 0.43 (0.30, 0.60)

nM³¹(Figure 4B), which closely paralleled both the human

mGluR5 K_i(0.9 nM) and the rat IVRO C_p,u-determined IC₅₀

(1.04 nM). The essentially identical human in vitro K_i, rat

IVRO C_p,u-derived IC₅₀, and NHP PET C_p,u-based IC₅₀

provided high conﬁdence in the interspecies translatability of

our in vitro screening assay.

Grounded in these well-deﬁned exposure−RO−response

correlations, we next wanted to establish a preclinical

exposure−RO−response relationship for 14 for PD-LID.

Thus, 14 was evaluated in MPTP-rendered Parkinsonian

NHP receiving ^L-DOPA-benserazide with well-characterized

consistent and reproducible dyskinesia; this preclinical model³²

has shown clinical translatability in PD-LID for the mGluR5

NAMs AFQ056^8b,9aand ADX48621.^8c,9bThis study was a

within-subject (n = 12), animal-handler-blinded design with

treatments pseudo-randomized in a Williams’ design including

both vehicle and positive control (amantadine, 20 mg/kg, po)

arms with ≥2 days between treatments based on each

compound’s t_1/2in NHP.³³A battery of behavioral measures

was assessed including activity, Parkinsonian disability, and

dyskinesia (composed of chorea and dystonia). On the basis of

satellite NHP pharmacokinetics data, three doses of 14 (0.1,

0.32, and 1.0 mg/kg, sc) were chosen to target maximal

projected mGlu5 RO of 77, 90, and 97%, respectively (Figure

5). Amantadine (p < 0.01) and 14 (0.32 and 1.0 mg/kg, p <

0.01 and p < 0.001, respectively) showed a statistically

The favorable potency, selectivity, and in vitro PK proﬁle of

14 warranted its PK evaluation in rats and NHP (Table 4). The

neuropharmacokinetics of 14 were determined in rats, where it

demonstrated net interneurocompartmental equilibrium and

parallel elimination rates in both central and peripheral

compartments. In rats and NHP, 14 had moderate systemic

blood clearance (CL_b) and a low-steady-state volume of

distribution (V_ss), which resulted in a relatively short half-life

(t_1/2). Acceptable oral bioavailability (F) was achieved in both

species.

We next examined the exposure−receptor occupancy (RO)

relationship of 14 in rats using an in vivo binding assay and in

NHP via positron emission tomography (PET). Rat in vivo RO

(IVRO) was determined using a modiﬁed [³H]3-methoxy-5-

pyridin-2-ylethynylpyridine ([³H]MPEPy) binding protocol.²⁸

The unbound plasma concentration of 14 (C_p,u) versus mGlu5

RO is shown in Figure 4A. Using a direct-response model, the

rat C_p,u-derived IC₅₀(95% conﬁdence interval) for 14 was 1.04

(0.94, 1.15) nM, consistent with its human mGluR5 K_i(0.9

nM).²⁹For determining RO in NHP (n = 2/dose, crossover

design), PET scans were taken using the mGluR5-selective

PET ligand [¹⁸F]FPEB with a vehicle (baseline scan) and four

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Figure 4. (A, B) mGlu5 RO vs unbound plasma 14 concentration (C_p,u) in rats and NHP, respectively. C) Coronal views of [¹⁸F]FPEB-dependent

PET images from a NHP receiving vehicle (baseline) and 14 (displacement, 0.0116 mg/kg iv bolus + 0.0180 mg/kg/h infusion over 120 min, 76%

RO).

po) and NHP (≤75 mg/kg/d, po) with no signiﬁcant clinical

or histopathological ﬁndings observed up to steady-state

AUC₀₋₂₄-based TI of 80 and 30, respectively (the TI

denominator is deﬁned as the species-speciﬁc total plasma

concentration of 14 aﬀording 80% mGlu5 RO over 24 h). On

the basis of this holistically favorable data set, 14 underwent 90-

day regulatory toxicology studies in rats (≤100 mg/kg/d, po)

and NHP (≤150 mg/kg/d, po). In rats, 14 showed no adverse

eﬀects at any tested dose (steady-state TI of 187 at 80 mg/kg/

d, po) during the study period. In NHP after 9 days of dosing,

erythematous skin lesions presented clinically in multiple body

regions in both the high- (n = 4/4, 150 mg/kg/day, TI of 460)

and mid- (n = 1/3, 50 mg/kg/day, TI of 150) dose males,

which terminated dosing in the high-dose male group. The

timing of clinical symptom onset coupled with the thorough

histopathological examination and histological immunostaining

features of multiple skin samples taken from all NHP after 90

days of dosing indicated a drug-dependent delayed-type

immune-mediated type IV hypersensitivity in these macaques.³⁴

This histopathology was detected in all dose groups,

irrespective of the clinical presentation of rash, aﬀording a TI

< 10. Thus, the preclinical development of 14 was terminated.

Signiﬁcantly, in a subsequent 14-day NHP ETS (n = 1/gender/

dose) with an mGluR5 NAM structurally distinct from 14 and

innocuous in a 14-day rat ETS (TI of 448 at the highest dose

tested), the identical NHP-speciﬁc microscopic ﬁndings were

observed in the mid-dose female (TI of 86).²⁹Collectively,

these data suggest this toxicity is potentially mechanism-, not

chemotype-, mediated. It is important to note that this speciﬁc

toxicity could be readily missed without performing a thorough

whole-body skin examination (both gross and histopatholog-

ical), as was the case with 14 in its 14-day NHP ETS because

the lack of visible skin rash masked the need for the

microscopic evaluation of skin samples, which are typically

not collected in exploratory toxicology studies. Thus, neither 14

or any other mGluR5 NAM has been progressed by Pﬁzer Inc.

to clinical trials.

○

Figure 5. Predicted mGlu5 RO vs time in NHP following 0.1 ( ),

□

●

0.32 ( ), and 1.0 ( ) mg/kg, sc, of 14. At each dose, an RO was

calculated by ﬁrst converting the mean total plasma 14 concentration

to C_p,uusing NHP f_u,p(0.064) and compound MW (332.4 ng/nmol)

and then incorporating C_p,u, I_max(100%), and IC₅₀(1.04 nM) into the

equation RO = (C_p,uI_max)/(C_p,u+ IC₅₀). Pharmacokinetic samples were

collected at 0.25, 0.5, 1, 2, 4, and 6 h postdose (n = 2/dose).

signiﬁcant reduction in dyskinesia without aﬀecting the L-

DOPA-mediated alleviation of Parkinsonism, as measured by

the disability score over the 6 h postdose evaluation period

(Figure 6). Holistically, these data showed that 14 modulates

excessive glutamate release in the basal ganglia and ameliorates

LID equivalent to amantadine at mGlu5 RO of 77 < x ≤ 90%,

with even greater attenuation at >90% RO.

In addition to its robust eﬃcacy in the PD-LID model, 14

also demonstrated suﬃcient safety and tolerability in 14-day

exploratory toxicology studies (ETS) in rats (≤80 mg/kg/d,

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Figure 6. Eﬀect of 14 (0.1, 0.32, and 1.0 mg/kg, sc) and amantadine (20 mg/kg, po) on the dyskinesia and total disability induced by L-DOPA

administration to MPTP-rendered Parkinsonian NHP. All groups were ﬁrst analyzed with a Friedman test followed by Dunn’s multiple comparison

test; ** p < 0.01 and *** p < 0.001, cf. vehicle + ^L-DOPA; n = 12 per treatment group. Individual animal scores as well as the median scores are

shown.

Pyrazolopyridine analogues 9−11 were synthesized in a

library format in which an advanced iodo-pyrazolopyridine

intermediate, 34, was employed to allow late-stage diversiﬁca-

tion on the aryl headgroup. As shown in Scheme 3,

condensation of aminopyrazole 29 with ethyl propiolate

resulted in ester 30, which, upon heating in acetic acid,

cyclized to form pyrazolopyridone 31. Subsequent chlorination

with phosphorus oxychloride yielded chloropyridine 32, which

was further iodinated to give key intermediate 34 with two

diﬀerentiated halogens as a synthetic handle for further

functionalization. For the exempliﬁed analogues 9−11,

pyridin-2-ylmethanol was used in a S_NAr reaction to yield

intermediate 34, which was converted to boronic ester 35 in

situ and subjected to Suzuki coupling to give desired analogues

9−11.

CHEMISTRY

■

As illustrated in Scheme 1, analogues 2−7 (Table 1) were

synthesized from a known pyrazolopyridine intermediate, 22,³⁵

a

Scheme 1

a

Reagents and conditions: (a) R₃OH, KOtBu, THF, rt; (b) TFA, rt;

(c) R₁R₂NH, HBTU, DIPEA, DMF, rt.

For the SAR study around the pyrazolopyrazine series, a

similar synthetic strategy was applied with an analogous chloro-,

iodo- pyrazolopyrazine intermediate, 41, targeted as a key

intermediate for late-stage diversiﬁcation (Scheme 4). Starting

from the same aminopyrazole, 29, oxidation with sodium nitrite

led to a nitroso intermediate 36. The conversion of 36 to

pyrazinone 37 was achieved via amine coupling with

diethylmalonate followed by cyclization under basic conditions

with the nitroso group to form desired pyrazinone 37. Ester

hydrolysis gave carboxylic acid 38, which then underwent

decarboxylation to aﬀord unsubstituted pyrazinone 39. A

similar chlorination and iodination sequence as described in

Scheme 3 aﬀorded key intermediate 41. S_NAr displacement

with pyridin-2-ylmethanol yielded pyridyl ether 42, which

underwent either Negishi coupling with aryl or heteroaryl

bromides or Suzuki coupling with aryl or heteroaryl boronic

acids to give desired analogues 12−19.

via a three-step sequence involving a S_NAr displacement

reaction followed by tert-butyl ester deprotection and amide

formation. These transformations were carried out either in

library format to provide variations in both the ether side chains

and the amides or in singleton fashion for the library hits

follow-up.

The synthesis of compound 8 is shown in Scheme 2. Starting

from dichloropyrazine 24, lithiation followed by trapping with

diethyloxalate aﬀorded ketoester 25. Condensation with methyl

hydrazine provided hydrazone 26, which, upon treatment with

sodium hydride, cyclized to give chloropyrazine ester

intermediate 27. Compound 8 was then prepared following a

similar three-step sequence as shown in Scheme 1 using

pyridin-2-ylmethanol for the S_NAr reaction and diethylamine

for the amide formation.

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a

Scheme 2

a

Reagents and conditions: (a) LiTMP, THF, −78 °C, diethyloxalate, 54%; (b) MeNHNH₂, 2 N HCl in Et₂O, EtOH, 80 °C; (c) NaH, THF, 0 °C to

rt, 71% over two steps; (d) pyridin-2-ylmethanol, KOtBu, THF, rt; (e) 1 N NaOH in H₂O, rt, 93% over two steps; (f) diethylamine, HBTU, DIPEA,

DMF, rt, 84%.

a

excellent mGluR5 potency and selectivity, favorable PK across

species, and a highly reproducible interspecies exposure−RO

relationship that could be quantitatively linked to eﬃcacy in a

translatable NHP model of PD-LID. However, the progression

of 14 to the clinic was terminated because of ﬁndings consistent

with a delayed-type immune-mediated type IV hypersensitivity

in a 90-day NHP regulatory toxicology study. Importantly, the

identical ﬁnding was also observed with a structurally distinct

mGluR5 NAM, implicating a potential mechanism linkage to

this immunotoxicity. Therefore, although there is encouraging

eﬃcacy (both preclinically and clinically) reported for mGluR5

NAMs in various indications, further eﬀorts are required to

determine deﬁnitively whether this NHP delayed-type immune-

mediated type IV hypersensitivity is indeed mechanism-based

and, consequently, highly relevant to patient safety in ongoing

(and future) mGluR5 NAM clinical trials.

Scheme 3

EXPERIMENTAL SECTION

■

a

Biology. Recombinant Human mGluR5 Radioligand Binding

Assay. Binding studies were performed in HEK-293FT cells

transiently transfected with the human mGluR5. Cells were

maintained in 293 FreeStyle Medium (Invitrogen) supplemented

with 10 mM HEPES and 50 μg/mL of G418 and transiently

transfected using polyethyleneimine. After 48 h, cells were centrifuged

two times at 3000g for 5 min at 4 °C in cold Dulbecco’s phosphate-

buﬀered saline. Membrane pellets were then frozen and stored at −80

°C until needed. Membrane concentration studies using [³H]MPEPy

determined that 0.5−2 mg of frozen cell paste per well was needed,

depending on the membrane preparation. A K_Dof 2 nM for

[³H]MPEPy was determined by saturation binding studies. For

inhibition binding concentration response studies, compounds were

prepared as 30 mM DMSO stocks and were then titrated in 100%

DMSO. Membrane paste was thawed, added to cold buﬀer containing

50 mM Tris with 10 mM EDTA at pH 7.2, homogenized with a

polytron, and then centrifuged at 40905g for 10 min at 4 °C. The

supernatant was discarded, the membrane pellet was resuspended in

cold buﬀer containing 50 mM Tris with 0.1 mM EDTA at pH 7.2, and

the suspension was homogenized with a polytron and then spun at

40905g for 10 min at 4 °C. The supernatant was discarded, and the

membrane pellet was resuspended in cold assay buﬀer containing 50

mM Tris at pH 7.2, which was homogenized with a polytron. The

volume was adjusted to provide 200 μL per well at the desired

membrane concentration. A 10× stock solution of [³H]MPEPy in

assay buﬀer was prepared, and the exact concentration was determined

by liquid scintillation counting. A ﬁnal assay concentration of

approximately 1 nM was used for inhibition binding experiments.

Reagents and conditions: (a) ethyl propiolate, NaOEt, EtOH, 40 °C,

90%; (b) AcOH, reﬂux; (c) POCl₃, 80 °C, 78% over two steps; (d)

NIS, triﬂuoroboric acid, CH₃CN, 80 °C, 88%; (e) pyridin-2-

ylmethanol, KOtBu, THF, rt, 69%; (f) 5,5,5′,5′-tetramethyl-2,2′-

bi(1,3,2-dioxaborinane), PdCl₂(dppf), KOAc, DMF, 80 °C; (g) ArBr,

PdCl₂(dppf), Na₂CO₃, DMF/H₂O (10:1), 80 °C.

For analogues bearing diﬀerent alkyl groups at the central

core pyrazole-N (20 and 21), an alternative synthetic route was

used (Scheme 5). Carbonylation of commercially available

bromopyridine 43 led to the formation of ester 44, which, upon

condensation with lithiated dichloropyrazine, aﬀorded ketone

45. Subsequent cyclization with alkyl hydrazines yielded

chloropyrazines 46a and 46b, which, upon S_NAr displacement

with pyridin-2-ylmethanol, aﬀorded desired analogues 20 and

21, respectively.

CONCLUSIONS

■

A novel series of alkyne-lacking pyrazolopyrazines was

identiﬁed as novel mGluR5 NAMs. The plasma instability

and low F associated with the original HTS hit were eﬃciently

addressed by focusing SAR eﬀorts on modulating physico-

chemical properties and replacing the metabolically labile amide

with bioisosteres. Compound 14 was identiﬁed as the best

molecule with a number of desirable attributes, including

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a

Scheme 4

a

Reagents and conditions: (a) NaNO₂, concentrated HCl, 0 °C to rt, 54%; (b) diethylmalonate, NaOEt, EtOH, reﬂux, 50%; (c) LiOH·H₂O, THF/

MeOH/H₂O (6:3:1), rt, 69%; (d) nitrobenzene, reﬂux, 86%; (e) POCl₃, H₃PO₄, DIPEA, reﬂux, 67%; (f) NIS, HBF₄, CH₃CN, reﬂux, 81%; (g)

pyridin-2-ylmethanol, KOtBu, THF, 0 °C, 96%; (h) ArBr, iPrMgBr/LiCl, THF rt, then ZnCl₂, 0 °C to rt, then PdCl₂(PPh₃)₂(20 mol %), 42, 80 °C;

(i) 5,5,5′,5′-tetramethyl-2,2′-bi(1,3,2-dioxaborinane), PdCl₂(dppf), KOAc, dioxane, 160 °C, 20 min, microwave; ArBr, PdCl₂(dppf), CH₂Cl₂,

Cs₂CO₃, dioxane/H₂O (10:1), 65 °C.

a

added to each well, and clear top plate seals were applied. After a

minimum 1 h incubation at rt, ﬁlter plates were read on a Trilux

(PerkinElmer) with a 1 min read per well. Percent eﬀects of

compound inhibition were determined using the DMSO and MPEP

control wells. IC₅₀values were determined with a logistic four-

parameter single-site model. K_ivalues were determined using the

Cheng−Prussof equation and the previously determined K_D. All

exempliﬁed active mGluR5 NAMs showed complete inhibition of

[³H]MPEPy in their concentration−response curves.

Scheme 5

mGluR5 and mGluR1-Mediated Inhibition of Calcium Flux in

HEK-293 Cells Expressing Rat mGluR5 or Human mGluR1 Using

FLIPR. HEK-293 cells stably expressing rat mGluR5 or human mGluR1

were maintained in Dulbecco’s modiﬁed Eagle medium with high

glucose (DMEM, Gibco) containing 10% fetal bovine serum

(HyClone). For FLIPR assays, 15000 cells per well were plated in

30 μL of glutamine-free DMEM (GIBCO) containing 5% FBS and 20

mM HEPES on Matrigel-coated or poly-^D-lysine-coated 384-well

black-wall clear-bottom polystyrene plates (Greiner). Plates were

incubated at 37 °C in 5% CO₂for 16−24 h. For antagonist evaluation

of test compounds, Fluo-8 calcium dye (HD Biosciences) was

prepared and added to the cell plates as above. After a 15 min

incubation at 37 °C in the dark, dye was removed, and 40 μL of

Hank’s balanced salt solution containing 1 mM probenecid was added

to each well. Five microliters of 9 times the ﬁnal concentration of test

compound was then added to each well using a Bravo (Agilent). Cell

plates were then incubated for 15 min at rt in the dark before being

placed in a FLIPR Tetra along with compound plates containing

glutamate at 5.5 times the ﬁnal assay concentration. Images were

collected before and after the transfer of 10 μL of glutamate per well

from the compound plate to the cell plate at rt for a total of 120

images. Excitation and emission wavelengths of 470−495 and 515−

575 were used, respectively. The glutamate challenge was adjusted for

each assay to produce an approximate EC₇₀response but was typically

between 320 and 1000 nM. The negative control was the glutamate

challenge, and the positive control was deﬁned by MPEP (10 μM).

Percent eﬀects of compound inhibition were determined using the

positive and negative control wells. IC₅₀values were determined with a

logistic four-parameter single-site-ﬁt model.

a

Reagents and conditions: (a) CO, Pd(OAc)₂, 1,1′-bis-

(diphenylphosphino)ferrocene, Et₃N, EtOH/DMF (2:1), 100 °C,

70%; (b) LiTMP, THF, −80 °C, 82%; (c) RNHNH₂, Et₃N, toluene,

rt; (d) pyridin-2-ylmethanol, KOtBu, THF, 0 °C.

Compounds were diluted into assay buﬀer at 10× ﬁnal assay

concentration in 25 μL per well. Nonspeciﬁc binding was determined

by the addition of MPEP to control wells at 10× of the ﬁnal assay

concentration of 10 μM. Negative control wells contained equivalent

concentrations of DMSO. To each assay well of a 96−well assay plate

was added 25 μL of compound or control, 25 μL of [³H]MPEPy stock

solution, and 200 μL of membrane stock solution. Plates were

incubated at rt for approximately 1 h. Similar results could be obtained

by incubation at 4 °C. A ﬁlter plate harvester (Packard) was used to

transfer well contents to PEI-coated GF/B ﬁlter plates (PerkinElmer)

that had been prewashed two times with wash buﬀer containing 50

mM Tris at pH 7.2. After transfer, three washes were performed with

wash buﬀer. Filter plates were allowed to dry overnight before bottom

plate seals were applied, 50 μL of Microscint 20 (PerkinElmer) was

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Rat in Vivo Receptor Occupancy (IVRO) Studies. Male Sprague−

Dawley rats (150−200 g; Charles River Laboratories) received 14 (2

mL/kg, sc) and [³H]MPEPy (30 μCi/kg, 1 mL/kg, iv) 60 and 1 min,

respectively, prior to euthanasia by rapid decapitation. The forebrain

(whole brain minus cerebellum) was rapidly dissected and

homogenized (3−5 s using a polytron at its highest setting) in 10

volumes of ice-cold assay buﬀer (10 mM K₂HPO₄and 100 mM KCl,

pH 7.4). Three replicates (400 μL) of the homogenate were ﬁltered

using a vacuum manifold ﬁltration system ﬁtted with 25 mm Whatman

GF/B ﬁlters soaked in assay buﬀer. The ﬁlters were immediately

washed (2 × 5 mL assay buﬀer) and transferred to scintillation vials,

where they soaked overnight in Ultima Gold (PerkinElmer) before

liquid scintillation counting. Total binding was determined by

measuring radioactivity in vehicle-treated animals, whereas nonspeciﬁc

binding was determined using animals treated with a saturating dose of

MPEP (50 mg/kg, sc).

brain atlas. Time-activity curves (TACs) were generated for ROIs such

as various cortical regions, putamen, caudate, and cerebellum and ﬁt to

a two-tissue (2T) compartment model using PMOD software (PMOD

2.85, PMOD Technologies) for determination of volume of

distribution V_t. Receptor occupancy (RO) was determined by analysis

of occupancy plots comparing the change in V_trelative to baseline

V_t.³⁷For all displacement scans, the mean total plasma concentration

of 14 during the 180 min scanning period was calculated by averaging

the exposure measured at 30, 120, and 210 min after the iv bolus of 14.

Chemistry. General Methods. Solvents and reagents were of

reagent grade and were used as supplied by the manufacturer. All

reactions were run under a N₂atmosphere. Organic extracts were

routinely dried over anhydrous Na₂SO₄or MgSO₄. Concentration

refers to rotary evaporation under reduced pressure. Chromatography

refers to ﬂash chromatography using disposable RediSepR_f4 to 120 g

silica columns or Biotage disposable columns on a CombiFlash

Companion or Biotage Horizon automatic puriﬁcation system.

Microwave reactions were carried out in a Smith Creator Personal

Chemistry microwave reactor. Puriﬁcation by mass-triggered HPLC

was carried out using Waters XTerra PrepMS C₁₈columns, 5 μm, 30 ×

100 mm steel. Compounds were presalted as TFA salts and diluted

with 1 mL of dimethyl sulfoxide. Samples were puriﬁed by mass-

triggered collection using a mobile phase of 0.1% triﬂuoroacetic acid in

water and acetonitrile with a starting gradient of 100% aqueous to

100% acetonitrile over 10 min at a 20 mL/min ﬂow rate. All target

compounds were analyzed using ultra-high-performance liquid

chromatography/ultraviolet/evaporative light-scattering detection

coupled to time-of-ﬂight mass spectrometry (UHPLC/UV/ELSD/

TOFMS). Unless otherwise noted, all tested compounds were found

to be ≥95% pure by this method.

UHPLC/MS Analysis. The UHPLC was performed on a Waters

ACQUITY UHPLC system (Waters, Milford, MA), which was

equipped with a binary solvent-delivery manager, column manager,

and sample manager coupled to ELSD and UV detectors (Waters,

Milford, MA). Detection was performed on a Waters LCT premier XE

mass spectrometer (Waters, Milford, MA). The instrument was ﬁtted

with an Acquity BEH (Bridged Ethane Hybrid) C₁₈column (30 mm ×

2.1 mm, 1.7 μm particle size, Waters, Milford, MA) operated at 60 °C.

Preparations of Analogues 2−7 in Library Format. Step 1:

synthesis of intermediate 23. To a stirred solution of R₃OH (2 equiv)

in THF under N₂at 23 °C was added potassium tert-butoxide (2

equiv). The reaction mixture was stirred at 23 °C for 20 min, and then

tert-butyl 6-ﬂuoro-1-methyl-1H-pyrazolo[3,4-b]pyridine-3-carboxylate

(22) (1 equiv) in THF was added. After 2 h, the reaction mixture was

concentrated in vacuo. The resulted thick brown oil was treated with a

large excess of triﬂuoro acetic acid, and the reaction mixture was

stirred at 23 °C for 18 h. The reaction mixture was concentrated in

vacuo and then triturated with methyl tert-butyl ether to give

carboxylic acid 23 with various side chains (OR₃).

Nonhuman Primate (NHP) Positron Emission Tomography (PET)

Studies. In NHP (Macaca fascicularis, 5 to 6 year old, n = 2/dose,

crossover design), PET scans with [¹⁸F]FPEB were taken on multiple

days at baseline and with various doses of 14. In preparation for PET

scanning, overnight-fasted NHPs were premedicated with ketamine

(10 mg/kg, IM) and glycopyrrolate (0.01 mg/kg, IM), intubated, and

maintained under isoﬂurane anesthesia (1.5−2.0% in oxygen) for the

duration of the scan procedure. An iv catheter was placed into the

saphenous vein for tracer injection and blood sampling. In addition, an

arterial access port was surgically implanted in the femoral artery for

assessing a plasma input function for PET data analysis. Dynamic PET

scans of 180 min in a Focus 220 microPET scanner (Siemens Medical

Systems) with [¹⁸F]FPEB were conducted on the 2 NHPs at both

baseline and drug displacement conditions. Each subject underwent

ﬁve PET scans: baseline and four doses (0.00136, 0.00273, 0.0116, or

0.02589 mg/kg iv bolus plus 0.00212, 0.00424, 0.0180, or 0.0402 mg/

kg/h infusion, respectively) of 14 targeting mGlu5 RO of 30, 50, 80,

and 90%, respectively. For displacement scans, 14 was administered

intravenously as an initial 5 min iv bolus at 30 min prior to [¹⁸F]FPEB

injection followed by a constant infusion for 205 min at the designated

dose levels to reach a stable total plasma concentration throughout the

PET scanning period. [¹⁸F]FPEB was synthesized in high speciﬁc

activity and radiochemical purity using a Siemens Eclipse cyclotron

and a GE FXn radiochemistry module. Before radiotracer injection, an

8 min transmission scan was acquired for attenuation and scatter

correction using a ⁵⁷Co point-source. [¹⁸F]FPEB was administered iv

as a bolus dose of approximately 1.0 mCi/kg in both baseline and

drug-displacement PET scans. For each displacement scan, venous

blood samples were collected at three time points (30, 120, and 210

min after the iv bolus of 14). Whole blood and plasma radioactivity

was measured by serial arterial blood sampling, and the percent parent

tracer was determined by HPLC. The physiological parameters of each

NHP, such as body temperature, respiration, and heart rate, were

monitored during the whole compound administration and PET

scanning period. PET image data were histogrammed into 25 frames

(ﬁve 1 min frames, ﬁve 5 min frames, and ﬁfteen 10 min frames) over

the 180 min dynamic scan duration. Three-dimensional (3D)

sinograms were created with corrections for random coincidences

and global dead time using a detector span of 3 and maximum ring

diﬀerence of 47 (the Focus 220 scanner consists of 48 rings of

detectors axially). Attenuation and scatter correction maps were

created from a measured ⁵⁷Co transmission scan (8 min duration).

Images were segmented into four attenuation factors (air, animal bed,

water, and bone) with 3D sinogram maps for attenuation and scatter

correction created from the segmented transmission image using

maximum a posteriori transmission reconstruction (MAP-TR). PET

images were reconstructed into a 256 × 256 × 95 volume with in-

plane pixel dimension of 0.9 mm and slice thickness of 0.8 mm with

corrections for detector normalization, decay, attenuation, and scatter

using fastMAP (maximum a posteriori) iterative image reconstruction

and inverse Fourier rebinning for fully 3D PET.³⁶Reconstructed

images were scaled to standardized uptake values (SUV). Brain regions

of interest (ROIs) were deﬁned for each subject on its baseline

[¹⁸F]FPEB scan by deformable registration from a segmented NHP

Step 2: amide coupling. Starting amines were obtained preweighed

in 2 dram vials (0.08 mmol, 1 equiv). A stock ﬁne suspension

containing the triﬂuoroacetic acid salt of crude template acid (23) (1

equiv), DIPEA (8 equiv), and HBTU (1.5 equiv) was prepared in dry

N,N-dimethylformamide (DMF). To the amines was added 0.5 mL of

the above suspension. The vials were capped and shaken at rt for 16 h.

Reactions were worked-up by addition of 1.5 mL of 1 N sodium

hydroxide solution and 2.5 mL of ethyl acetate. Samples were

centrifuged to break up emulsions. Using a Tecan workstation, the

organic layers were extracted and loaded onto an SPE cartridge

charged with Na₂SO₄(6 mL, ∼1 g). The extraction was repeated two

times. The combined organics were concentrated in a Genevac. Dry

samples were dissolved in 1 mL of dimethyl sulfoxide, ﬁltered, and

submitted directly to mass-triggered HPLC puriﬁcation. The

compounds were characterized by UHPLC and MS. For analogues

(3−6) that were followed-up with resynthesis, their ¹H NMR data are

also shown below.

N-Ethyl-1-methyl-6-((6-methylpyridin-2-yl)methoxy)-1H-

pyrazolo[3,4-b]pyridine-3-carboxamide (2). The title compound was

prepared using (6-methylpyridin-2-yl)methanol and ethyl amine. MS

(ES⁺): m/z 326.2 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

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N,N-Diethyl-1-methyl-6-((6-methylpyridin-2-yl)methoxy)-1H-

pyrazolo[3,4-b]pyridine-3-carboxamide (3). The title compound was

prepared using (6-methylpyridin-2-yl)methanol and diethyl amine

(45% yield over two steps in resynthesis). ¹H NMR (400 MHz,

DMSO-d₆) δ 8.30 (d, J = 8.6 Hz, 1 H), 8.18 (t, J = 8.2 Hz, 1H), 7.77

(d, J = 6.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H),

5.70 (s, 2H), 4.00 (s, 3H), 3.79 (q, J = 7.1 Hz, 2H), 3.48 (q, J = 7.1

Hz, 2H), 2.68 (s, 3H), 1.18 (dt, J = 10.6, 6.9 Hz, 6H). MS (ES⁺): m/z

354.2 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

heated to 80 °C for 2 h. It was then cooled, poured into saturated

aqueous NaHCO₃solution, and washed with ethyl acetate (2×). The

organic layers were combined, dried over MgSO₄, and concentrated in

vacuo to give 26, which was used without further puriﬁcation. MS

(ES⁺): m/z 276.1 (M + 1). GCMS 100%.

Step 3: synthesis of ethyl 6-chloro-1-methyl-1H-pyrazolo[3,4-

b]pyrazine-3-carboxylate (27). To a stirred solution of ethyl 2-(3,5-

dichloropyrazin-2-yl)-2-(2-methylhydrazono)acetate (26) from the

previous step (theoretical 156 mmol) in 400 mL of THF at 0 °C

was added sodium hydride (60% dispersion in mineral oil, 8.75 g, 219

mmol) slowly in portions. The reaction mixture was warmed to 23 °C

for 30 min. It was quenched with saturated ammonium chloride

solution and then poured into saturated ammonium chloride solution.

The pH of the aqueous layer was adjusted to 5 with the addition of 1

N hydrochloride solution, and it was then washed with dichloro-

methane. The organic layers were combined, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed using silica gel

chromatography with 5% ethyl acetate/dichloromethane as eluent

followed by partial concentration from dichloromethane/heptanes to

give 27 as an oﬀ-white solid in 71% yield. ¹H NMR (400 MHz,

CDCl₃) δ 8.74 (s, 1H), 4.59 (q, J = 7.0 Hz, 2H), 4.24 (s, 3H), 1.49 (t,

J = 7.1 Hz, 3H). MS (ES⁺): m/z 241.1 (M + 1). UHPLC: 100% (UV),

100% (ELSD).

N,N-Diethyl-1-methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-

b]pyridine-3-carboxamide (4). The title compound was prepared

using pyridin-2-yl methanol and diethylamine (71% yield over two

1

steps in resynthesis). H NMR (400 MHz, CD₃OD) δ 8.53 (dt, J =

4.9, 0.8 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H), 7.85 (td, J = 7.8, 1.7 Hz,

1H), 7.61 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.5, 5.0 Hz, 1H), 6.83 (d, J

= 8.6 Hz, 1H), 5.58 (s, 2H), 4.00 (s, 3H), 3.85 (q, J = 7.1 Hz, 2H),

3.58 (q, J = 6.9 Hz, 2H), 1.27 (q, J = 6.7 Hz, 6H). MS (ES⁺): m/z

340.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

(R)-(1-Methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyridin-

3-yl)(2-methylpyrrolidin-1-yl)methanone (5). The title compound

was prepared using pyridin-2-yl methanol and (R)-2-methylpyrrolidine

1

(91% yield over two steps in resynthesis). H NMR as 2:1 mixture of

rotamers (400 MHz, DMSO-d₆) δ 8.54−8.62 (m, 1H), 8.32−8.41 (m,

1H), 7.83 (td, J = 7.7, 2.0 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.35 (ddd,

J = 7.6, 4.9, 1.2 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 5.56 (s, 2H), 4.94−

5.06 (m, 0.35H), 4.23−4.35 (m, 0.7H), 3.97−4.06 (m, 3.7H), 3.86−

3.96 (m, 0.7H), 3.53−3.62 (m, 0.7H), 1.91−2.10 (m, 2H), 1.80−1.91

(m, 1H), 1.70 (dd, J = 11.3, 5.5 Hz, 1H), 1.51−1.61 (m, 1H), 1.24 (d,

J = 6.2 Hz, 2H), 1.17 (d, J = 6.2 Hz, 1H). MS (ES⁺): m/z 352.2 (M +

1). UHPLC: 100% (UV), 100% (ELSD).

Step 4: synthesis of 1-methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-

[3,4-b]pyrazine-3-carboxylic acid (28). To a stirred solution of

potassium tert-butoxide (11 g, 94 mmol) in 100 mL of THF under

N₂at 23 °C was added 2-pyridyl carbinol (9.5 mL, 97 mmol). The

mixture was stirred at 23 °C for 20 min. The resulting suspension was

added by fast drops to ethyl 6-chloro-1-methyl-1H-pyrazolo[3,4-

b]pyrazine-3-carboxylate (27) (15.4 g, 64 mmol) partially dissolved in

200 mL of THF at 0 °C. After the addition was complete in 10 min,

300 mL of 1 N sodium hydroxide solution was added. The reaction

mixture was removed from the cold bath and stirred at 23 °C for 2 h. It

was then concentrated in vacuo to remove THF and was then chilled

in an ice/water bath. The pH was adjusted to 4 by the addition of 200

mL of a 1 N hydrochloride solution. The tan solid that precipitated

was collected by vacuum ﬁltration and washed with chilled water to

(1-Methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyridin-3-

yl)(morpholino)methanone (6). The title compound was prepared

using pyridin-2-yl methanol and morpholine (64% yield over two steps

1

in resynthesis). H NMR (400 MHz, CDCl₃) δ 8.63 (d, J = 4.3 Hz,

1H), 8.35 (d, J = 8.6 Hz, 1H), 7.72 (td, J = 7.7, 1.8 Hz, 1H), 7.51 (d, J

= 7.8 Hz, 1H), 7.24 (dd, J = 7.1, 5.2 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H),

5.64 (s, 2H), 4.29 (br s, 2H) 4.02 (s, 3H) 3.72−3.90 (m, 6H). m/z

354.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

1

give 16.9 g of 28 in 93% yield over two steps. H NMR (400 MHz,

1-Methyl-N-((3-methylisoxazol-5-yl)methyl)-6-(pyridin-2-ylme-

thoxy)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (7). The title

compound was prepared in library format using pyridin-2-yl methanol

and (3-methylisoxazol-5-yl)methyl amine. MS (ES⁺): m/z 379.2 (M +

1). UHPLC: 100% (UV), 99% (ELSD).

CD₃OD) δ 8.58−8.67 (m, 1H), 8.45 (s, 1H), 8.04 (td, J = 7.8, 1.8 Hz,

1H), 7.79 (d, J = 7.8 Hz, 1H), 7.53 (ddd, J = 7.6, 5.1, 1.2 Hz, 1H), 5.70

(s, 2H), 4.08 (s, 3H). MS (ES⁺): m/z 286.1 (M + 1). UHPLC: 85%

(UV), 81% (ELSD).

Step 5: synthesis of N,N-diethyl-1-methyl-6-(pyridin-2-ylmethoxy)-

1H-pyrazolo[3,4-b]pyrazine-3-carboxamide (8). To a suspension of 1-

methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine-3-carbox-

ylic acid (28) (16.9 g, 59 mmol) in 198 mL of DMF at 23 °C was

added di-isopropyl ethyl amine (42 mL, 240 mmol), O-(benzotriazol-

1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuorophosphate (25 g, 66

mmol), and diethyl amine (6.9 mL, 65 mmol). The reaction mixture

was stirred 1 h at 23 °C. It was then poured into saturated sodium

bicarbonate solution and extracted with ethyl acetate (1×) and

dichloromethane (2×). The organic layers were combined, washed

with a 1 N lithium chloride solution, and then concentrated to

approximately half-volume. They were then washed twice with a 1 N

hydrochloride solution. The acidic extracts were carefully neutralized

to pH 5 with a 6 N sodium hydroxide solution and were then extracted

with dichloromethane. These organic extracts were dried over MgSO₄,

ﬁltered, and concentrated in vacuo. The crude material was crystallized

twice from hot acetonitrile to give 8 in 84% yield. ¹H NMR (400 MHz,

CDCl₃) δ 8.65 (d, J = 4.1 Hz, 1H), 7.75 (td, J = 7.7, 1.8 Hz, 1H), 7.52

(d, J = 7.8 Hz, 1H), 7.28 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 5.63 (s, 2H),

4.04 (s, 3H), 3.65 (q, J = 7.2 Hz, 2H), 3.56 (q, J = 7.0 Hz, 2H), 1.32 (t,

J = 7.1 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H). MS (ES⁺): m/z 341.2 (M +

1). UHPLC: 89% (UV), 100% (ELSD). The hydrochloride salt of 8

was prepared by dissolving the free base in methanol, adding 2 M

hydrochloride in ethyl ether, and concentrating to a slurry. The oﬀ-

white crystals were collected by vacuum ﬁltration. ¹H NMR (400

MHz, CDCl₃) δ 8.92 (ddd, J = 5.9, 1.6, 0.8 Hz, 1H), 8.72 (td, J = 7.9,

1.6 Hz, 1H), 8.46 (s, 1H), 8.35 (d, J = 8.2 Hz, 1H), 8.12 (t, J = 6.9 Hz,

1H), 5.97 (s, 2H), 4.05 (s, 3H), 3.64 (q, J = 7.1 Hz, 2H), 3.55 (q, J =

Preparation of N,N-Diethyl-1-methyl-6-(pyridin-2-ylmethoxy)-

1H-pyrazolo[3,4-b]pyrazine-3-carboxamide (8). Step 1: synthesis of

ethyl 2-(3,5-dichloropyrazin-2-yl)-2-oxoacetate (25). To a stirred

solution of tetramethyl piperidine (28 mL, 160 mmol) under N₂at

−40 °C was added n-BuLi (2.5 M in hexanes, 79 mL, 198 mmol)

dropwise. The solution was stirred cold for 20 min. To a cooled (−73

°C, internal thermometer) solution of 2,6-dichloropyrazine (24) (25 g,

160 mmol) and diethyl oxalate (25 g, 170 mmol) in 500 mL of THF

under N₂was added the freshly prepared lithium tetramethyl

piperidine solution dropwise so as to maintain the −73 °C internal

temperature. The reaction was quenched cold 15 min after the

addition was complete by the dropwise addition of acetic acid (9.6 mL,

160 mmol). The reaction mixture was poured into saturated

ammonium chloride solution and washed with ethyl acetate (3×).

The organic layers were combined, dried over magnesium sulfate, and

concentrated in vacuo. The residue was puriﬁed using silica gel

chromatography with a 0−50% ethyl acetate/heptane gradient as

eluent to aﬀord 25 as an orange oil in 54% yield. ¹H NMR (400 MHz,

CDCl₃, two rotamers) δ 8.61 (s, 1H), 8.50 (s, 1H), 4.45 (q, J = 7.1 Hz,

2H), 4.33 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.1

Hz, 3H). MS (ES⁺): m/z 249.9 (M + 1). GCMS: 100%.

Step 2: synthesis of ethyl 2-(3,5-dichloropyrazin-2-yl)-2-(2-

methylhydrazono)acetate (26). To a solution of methyl hydrazine

(8.6 mL, 164 mmol) in 90 mL of ethanol at 0 °C was added

hydrochloride (2 M in ethyl ether, 82 mL, 164 mmol) dropwise. Ethyl

2-(3,5-dichloropyrazin-2-yl)-2-oxoacetate (25) (38.9 g, 156 mmol) in

90 mL of ethanol was then added dropwise. The reaction mixture was

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7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H). MS

(ES⁺): m/z 341.2 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

Preparation of 3-Iodo-1-methyl-6-(pyridin-2-ylmethoxy)-1H-

pyrazolo[3,4-b]pyridine (34). Step 1: synthesis of ethyl 3-ethoxy-3-

((1-methyl-1H-pyrazol-5-yl)amino)propanoate (30). Sodium metal

(3.84 g, 166 mmol) was dissolved in ethanol (120 mL) at 40 °C under

N₂. 1-Methyl-1H-pyrazol-5-amine (29) (4.04 g, 41.6 mmol) was then

added in one portion. The reaction mixture was stirred for 1 h, and

then ethyl propiolate (8.16 g, 83 mmol) was added dropwise over 20

min. Upon completion of addition, the reaction mixture was heated to

reﬂux over 16 h. The reaction was then cooled to rt and concentrated

in vacuo. The residue was partitioned between water (200 mL) and

ethyl acetate (200 mL). The aqueous layer was further extracted with

ethyl acetate (3 × 100 mL). The organic layers were combined,washed

with water and brine, and dried over MgSO₄. The solvent was

removed in vacuo, and the residue was triturated with petroleum ether

to give 9.08 g of 30 as a light brown solid in 90% yield. ¹H NMR (400

MHz, CDCl₃) δ 8.43 (br s, 1H), 7.37 (d, J = 1.8 Hz, 1H), 6.24 (d, J =

2.0 Hz, 1H), 4.80 (t, J = 4.7 Hz, 1H), 3.72 (m, 2H), 3.69 (s, 3H), 3.57

(m, 2H), 2.73 (d, J = 4.5 Hz, 2H), 1.22 (t, J = 7.0 Hz, 6H). MS (ES⁻):

m/z 240.2 (M − 1).

Step 2: synthesis of 1-methyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one

(31). 3-Ethoxy-3-((1-methyl-1H-pyrazol-5-yl)amino)propanoate (30)

(9.08 g, 37.6 mmol) was dissolved in acetic acid (125 mL) in a round-

bottomed ﬂask equipped with a condenser at rt. The reaction mixture

was then heated to reﬂux for 16 h. The reaction was then cooled to rt,

and the excess acetic acid was removed in vacuo. The resulting brown

solid was further triturated with petroleum ether (2×) and dried in

vacuo to give 31 as a light brown solid, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J

= 9.4 Hz, 1H), 7.66 (s, 1H), 6.30 (d, J = 9.2 Hz, 1H), 3.96 (s, 3H). MS

(ES⁺): m/z 150.0 (M + 1).

Step 3: synthesis of 6-chloro-1-methyl-1H-pyrazolo[3,4-b]pyridine

(32). 1-Methyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (31) from the

previous step was suspended in phosphorus oxychloride (34 mL, 376

mmol), and the reaction mixture was heated at 80 °C under N₂for 16

h. The reaction mixture was quenched by pouring it into a vigorously

stirring mixture of ice water and diethyl ether. After stirring for 40 min,

the organic layer was separated, and the aqueous layer was extracted

with dichloromethane. The combined organic layers were dried over

Na₂SO₄, ﬁltered, and concentrated. Trituration of the residue with

petroleum ether aﬀorded 4.9 g of 32 as a white solid in 78% yield over

two steps. ¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (t, J = 4.2 Hz, 2H),

7.08 (d, J = 8.4 Hz, 1H), 4.08 (s, 3H). MS (ES⁺): m/z 168.1 (M+1).

Step 4: synthesis of 6-chloro-3-iodo-1-methyl-1H-pyrazolo[3,4-

b]pyridine (33). To a solution of 6-chloro-1-methyl-1H-pyrazolo-

[3,4-b]pyridine (32) (4.65 g, 27.7 mmol) in acetonitrile (90 mL) was

added N-iodosuccinimide (6.44 g, 27.7 mmol) followed by 48%

aqueous tetraﬂuoroboric acid (17.4 mL, 139 mmol). The reaction

mixture was heated at 80 °C for 1 h and was then poured into

saturated aqueous sodium bicarbonate solution and stirred for 30 min.

The resulting precipitate was ﬁltered, and the collected solid was

washed with saturated aqueous sodium thiosulfate solution and then

with water. Trituration with n-pentane provided 7.2 g of 33 as a cream

colored solid in 88% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 7.94 (d,

J = 8.2 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H). MS (ES⁺): m/z

294.0 (M+1).

Step 5: synthesis of 3-iodo-1-methyl-6-(pyridin-2-ylmethoxy)-1H-

pyrazolo[3,4-b]pyridine (34). To a stirred solution of pyridin-2-

ylmethanol (1.97 mL, 20.4 mmol) in DMSO under N₂at rt was added

potassium tert-butoxide (2.3 g, 20.4 mmol). The reaction mixture was

stirred for 30 min, and then 6-chloro-3-iodo-1-methyl-1H-pyrazolo-

[3,4-b]pyridine (33) (5.0 g, 17.0 mmol) was added. The reaction

mixture was stirred for additional 20 min, diluted with water (250

mL), and extracted with ethyl acetate (3 × 150 mL). The combined

organic extracts were dried over MgSO₄, ﬁltered, and concentrated in

vacuo. The residue was puriﬁed using silica gel chromatography with a

0−60% ethyl acetate/heptane gradient as eluent to aﬀord 4.33 g of 34

as an oﬀ-white solid in 69% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.58

(d, J = 4.9 Hz, 1H), 7.67 (m, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.45 (d, J

= 7.8 Hz, 1H), 7.20 (m, 1H), 6.70 (d, J = 8.6 Hz, 1H), 5.57 (s, 2H),

3.95 (s, 3H). MS (ES⁺): m/z 366.9 (M + 1).

Preparation of Analogues 9−11 in Library Format. Step 1:

synthesis of 1-methyl-6-(pyridin-2-ylmethoxy)-3-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (35). Potassium

acetate (1.60 g, 16.4 mmol) was combined with 4,4,4′,4′,5,5,5′,5′-

octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.54 g, 6.0 mmol), 3-iodo-1-

methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyridine (34)

(2.00 g, 5.5 mmol), and [1,1′ -bis(diphenylphosphino)ferrocene]-

dichloropalladium(II) (117 mg, 0.16 mmol). The mixture was

subjected to three cycles of vacuum evacuation followed by ﬁlling

with nitrogen, and the vial was then charged with degassed DMF (25

mL). After vigorous stirring at rt to provide a homogeneous mixture,

the reaction mixture was heated at 80 °C for 1 h, ﬁltered through a

plug of Celite, and washed with dichloromethane. The ﬁltrate was

concentrated in vacuo to provide corresponding boronic ester 35 as a

crude residue, which was used in the following step in library format

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 8.64 (d, J

= 4.1 Hz, 1H), 7.73 (td, J = 7.6, 1.7 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H),

7.51 (d, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.6, 2.7 Hz, 1H), 6.77 (d, J = 8.8

Hz, 1H), 5.63 (s, 2H), 4.02 (s, 3H), 1.28 (s, 6H), 1.26 (s, 6H). MS

(ES⁺): m/z 367.0 (M + 1).

Step 2: library synthesis via Suzuki coupling. Boronic ester residue

35 was combined with sodium carbonate (0.26 mmol), tetrakis-

(triphenylphosphine)palladium(0) (0.0016 mmol), and the appro-

priate aryl or heteroaryl bromide or iodide (0.10 mmol) in DMF (1

mL). The reaction mixture was degassed using two rounds of vacuum

evacuation followed by nitrogen ﬁll. The reaction mixture was shaken

and heated at 80 °C for 20 h. It was then partitioned between water

(1.5 mL) and ethyl acetate (2.5 mL) and vortexed, using centrifugation

to break up any emulsions. The organic layer was passed through a

solid-phase extraction cartridge ﬁlled with Na₂SO₄(approximately 1

g). This extraction procedure was carried out a total of three times,

and the combined ﬁltrates were concentrated and puriﬁed via reversed-

phase HPLC. The compounds were characterized by UHPLC and MS.

For analogues (10 and 11) that were followed-up with resynthesis,

1

their H NMR data are also shown below.

1-Methyl-3-phenyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]-

pyridine (9). The title compound was prepared using iodobenzene.

MS (ES⁺): m/z 317.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

1-Methyl-3-(pyridin-2-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-

[3,4-b]pyridine (10). The title compound was prepared using 2-bromo

1

pyridine (47% yield in resynthesis). H NMR (400 MHz, CDCl₃) δ

8.75 (d, J = 8.6 Hz, 1H), 8.69 (m, 1H), 8.63 (m, 1H), 8.12 (dt, J = 7.8,

1.0 Hz, 1H), 7.70−7.76 (m, 2H), 7.53 (d, J = 7.8 Hz, 1H), 7.22−7.24

(m, 2H), 6.82 (d, J = 8.8 Hz, 1H), 5.65 (s, 2H), 4.07 (s, 3H). MS

(ES⁺): m/z 318.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

1-Methyl-6-(pyridin-2-ylmethoxy)-3-(pyridin-3-yl)-1H-pyrazolo-

[3,4-b]pyridine (11). The title compound was prepared using 3-bromo

1

pyridine (36% yield in resynthesis). H NMR (400 MHz, CDCl₃) δ

9.17 (br s, 1H), 8.65 (m, 1H), 8.24 (dt, J = 8.1, 1.8 Hz, 1H), 8.18 (d, J

= 8.8 Hz, 1H), 7.74 (td, J = 7.8, 1.7 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H),

7.42 (dd, J = 7.8, 4.8 Hz, 1H), 7.26 (dd, J = 8.1, 5.2 Hz, 1H), 6.83 (d, J

= 8.6 Hz, 1H), 5.67 (s, 2H), 4.08 (s, 3H). MS (ES⁺): m/z 318.1 (M +

1). UHPLC: 100% (UV), 100% (ELSD).

Preparation of 3-Iodo-1-methyl-6-(pyridin-2-ylmethoxy)-1H-

pyrazolo[3,4-b]pyrazine (42). Step 1: synthesis of 1-methyl-4-

nitroso-1H-pyrazol-5-amine (36). Concentrated hydrochloric acid

(160 mL) was added to a solution of 1-methyl-1H-pyrazol-5-amine

(29) (200 g, 2.06 mol) in water (1.4 L), and the mixture was cooled to

0 °C. A solution of sodium nitrite (127.7 g, 1.851 mol) in water (400

mL) was added dropwise while maintaining the temperature below 5

°C, and the reaction was allowed to stir for an additional 2 h at rt. The

yellow solid that precipitated during the reaction was isolated via

ﬁltration and was washed with cold water to aﬀord 140 g of 36 as a

brown solid in 54% yield. This was used in the next step without

additional puriﬁcation. MS (ES⁺): m/z 127.1 (M + 1). UHPLC: 100%

(UV), 100% (ELSD). R_f= 0.4 via thin-layer chromatography (TLC)

(eluent = 10% methanol in dichloromethane).

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Step 2: synthesis of ethyl 1-methyl-6-oxo-6,7-dihydro-1H-pyrazolo-

[3,4-b]pyrazine-5-carboxylate (37). Sodium metal (52.0 g, 2.26 mol)

was allowed to react with ethanol (2 L), diethyl malonate (380 mL,

2.50 mol) was added dropwise, and the reaction mixture was stirred

for 30 min at rt. 1-Methyl-4-nitroso-1H-pyrazol-5-amine (36) (285 g,

2.26 mol), prepared as described in step 1, was added, and the reaction

mixture was heated to reﬂux for 18 h. Removal of solvent in vacuo

provided a residue, which was triturated with petroleum ether and

ﬁltered. The resulting solid was dissolved in water, and the pH of the

solution was adjusted to a value of 3 to 4 by addition of dilute aqueous

hydrochloric acid. The resulting precipitate was isolated via ﬁltration

and triturated with n-pentane to aﬀord 250 g of 37 as a yellow solid in

50% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 8.29 (br s, 1H), 4.35 (q,

J = 7.1 Hz, 2H), 3.93 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). MS (ES⁺): m/z

223.1 (M + 1)

74.8 mmol) in THF (170 mL) at 0 °C at a rate that maintained the

internal reaction temperature below 10 °C. The reaction mixture was

stirred for 10 min and was then diluted with water (340 mL) and

concentrated in vacuo. The aqueous residue was extracted twice with

dichloromethane, and the combined organic extracts were dried over

MgSO₄, ﬁltered, and concentrated in vacuo. The residue was triturated

with cold ethyl acetate to aﬀord 24.1 g of 42 as a white solid in 96%

1

yield. H NMR (400 MHz, CDCl₃) δ 8.65 (ddd, J = 4.9, 1.8, 0.9 Hz,

1H), 8.31 (s, 1H), 7.76 (ddd, J = 7 0.8, 7.6, 1.8 Hz, 1H), 7.52 (br d, J =

7.9 Hz, 1H), 7.27−7.31 (m, 1H), 5.63 (s, 2H), 4.05 (s, 3H). MS

(ES⁺): m/z 367.9 (M + 1).

Preparation of Analogues 12−19 from Advanced Intermediate

42. Method A (Negishi Coupling, Singleton format). A solution of

isopropylmagnesium chloride/lithium chloride complex (1.3 M in

THF, 2.6 equiv) was added dropwise to a −78 °C solution of the

appropriate heteroaryl bromide in THF (2.0 equiv), and the mixture

was stirred for 20 min. Zinc chloride (0.5 M solution in THF, 2.6

equiv) was added dropwise; after 10 min, the cooling bath was

removed, and the reaction mixture was allowed to stir at rt for 10 min.

A mixture of 3-iodo-1-methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-

[3,4-b]pyrazine (42) (1 equiv) and dichlorobis(triphenylphosphine)-

palladium(II) (0.2 equiv) was added, and the reaction mixture was

immersed in an 80 °C oil bath for 1.5 h. After cooling to rt, the

reaction mixture was diluted with ethyl acetate, quenched with a 1 N

aqueous sodium hydroxide solution, and then extracted twice with

ethyl acetate. The combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. Two puriﬁcations using silica gel

chromatography (gradients: 0−6% methanol in dichloromethane

followed by 0−3% methanol in dichloromethane) aﬀorded the desired

analogues.

Method B (Suzuki Coupling, Library format). Potassium acetate (4

equiv) was dried under vacuum and then combined in a microwave

vial with 5,5,5′,5′-tetramethyl-2,2′-bi-1,3,2-dioxaborinane (1.7 equiv),

3-iodo-1-methyl-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine

(42) (1.0 equiv), and [1,1′ -bis(diphenylphosphino)ferrocene]-

dichloropalladium(II) (0.2 equiv). The mixture was subjected to

three cycles of vacuum evacuation followed by ﬁlling with nitrogen,

and the vial was then charged with degassed 1,4-dioxane. After

vigorous stirring at rt to provide a homogeneous mixture, the reaction

mixture was heated at 160 °C for 35 min under microwave irradiation,

ﬁltered through a plug of Celite, and washed with dichloromethane.

The ﬁltrate was concentrated in vacuo to provide the corresponding

boronic ester as a crude residue, which was used in the following step

in library format without further puriﬁcation. The boronic ester

residue from the previous step was combined with cesium ﬂuoride

(0.17 mmol), tetrakis(triphenylphosphine)palladium(0) (0.0034

mmol), and the appropriate heteroaryl bromide (0.10 mmol) in 1,4-

dioxane (1 mL). The reaction mixture was degassed using two rounds

of vacuum evacuation followed by nitrogen ﬁll. The reaction mixture

was shaken and heated at 90 °C for 18 h. It was then partitioned

between water (1.5 mL) and ethyl acetate (2.5 mL) and vortexed,

using centrifugation to break up any emulsions. The organic layer was

passed through a solid-phase extraction cartridge ﬁlled with Na₂SO₄

(approximately 1 g). This extraction procedure was carried out a total

of three times, and the combined ﬁltrates were concentrated and

puriﬁed via reversed-phase HPLC. The compounds were characterized

by UHPLC and MS.

Step 3: synthesis of 1-methyl-6-oxo-6,7-dihydro-1H-pyrazolo[3,4-

b]pyrazine-5-carboxylic acid (38). Ethyl 1-methyl-6-oxo-6,7-dihydro-

1H-pyrazolo[3,4-b]pyrazine-5-carboxylate (37) (250 g, 1.12 mol) was

partially dissolved in a mixture of THF/methanol/water (6:3:1 ratio,

5.4 L). Lithium hydroxide monohydrate (330 g, 7.86 mol) was added

at rt, and the reaction mixture became homogeneous. After the

reaction mixture had stirred for 4 h at rt and produced a tan

precipitate, the reaction mixture was diluted with ethyl acetate and

ﬁltered. The isolated solid was washed with EtOAc and partially

dissolved in a minimum quantity of water. After adjustment of the pH

to approximately 2 via addition of dilute aqueous hydrochloric acid,

the solid was ﬁltered and triturated with n-pentane to aﬀord 150 g of

1

38 as a light brown solid in 69% yield. H NMR (300 MHz, DMSO-

d₆) δ 8.30 (s, 1H), 3.92 (s, 3H). MS (ES⁺): m/z 193.1 (M − 1).

Step 4: synthesis of 1-methyl-1H-pyrazolo[3,4-b]pyrazin-6(7H)-one

(39). 1-Methyl-6-oxo-6,7-dihydro-1H-pyrazolo[3,4-b]pyrazine-5-car-

boxylic acid (38) (150 g, 0.773 mol) was suspended in nitrobenzene

(1.2 L) and heated to reﬂux for 6 h. After the reaction mixture cooled

to rt, it was diluted with petroleum ether and stirred for 30 min; the

resulting precipitate was isolated via ﬁltration, and the solid was

washed with petroleum ether. Trituration with n-pentane provided 100

g of 39 as a brown solid in 86% yield. ¹H NMR (300 MHz, DMSO-d₆)

δ 12.5 (br s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 3.90 (s, 3H). MS (ES⁺):

m/z 149.1 (M − 1).

Step 5: synthesis of 6-chloro-1-methyl-1H-pyrazolo[3,4-b]pyrazine

(40). 1-Methyl-1,7-dihydro-6H-pyrazolo[3,4-b]pyrazin-6-one (39)

(100 g, 666 mmol) was suspended in phosphorus oxychloride (306

mL, 3.28 mol). Phosphoric acid (10 mL) was added dropwise over 5

min at rt followed by addition of N,N-diisopropylethylamine (116 mL,

666 mmol) dropwise over 30 min. The reaction mixture was heated to

reﬂux for 16 h and was then poured into a vigorously stirring mixture

of ice water and diethyl ether. After stirring for 40 min, the organic

layer was separated, and the aqueous layer was extracted with

dichloromethane. The combined organic layers were dried over

Na₂SO₄, ﬁltered, and concentrated. Trituration of the residue with n-

1

pentane aﬀorded 75 g of 40 as a light orange solid in 67% yield. H

NMR (400 MHz, DMSO-d₆) δ 8.72 (s, 1H), 8.52 (s, 1H), 4.06 (s,

3H). MS (ES⁺): m/z 169.1 (M + 1).

Step 6: synthesis of 6-chloro-3-iodo-1-methyl-1H-pyrazolo[3,4-

b]pyrazine (41). To a solution of 6-chloro-1-methyl-1H-pyrazolo-

[3,4-b]pyrazine (40) (50.0 g, 296 mmol) in acetonitrile (500 mL) was

added N-iodosuccinimide (66.7 g, 296 mmol) followed by 48%

aqueous tetraﬂuoroboric acid (93.2 mL, 713 mmol). The reaction

mixture was heated to reﬂux for 2 h and was then poured into a

saturated aqueous sodium bicarbonate solution and stirred for 30 min.

The resulting precipitate was ﬁltered; the solid was washed with

saturated aqueous sodium thiosulfate solution and then with water.

Trituration with n-pentane provided 72 g of 41 as a yellow solid in

1-Methyl-6-(pyridin-2-ylmethoxy)-3-(pyridin-3-yl)-1H-pyrazolo-

[3,4-b]pyrazine (12). The title compound was prepared using method

B (library format) with 3-bromopyridine. MS (ES⁺): m/z 319.2 (M +

1). UHPLC: 100% (UV), 100% (ELSD).

1-Methyl-3-(3-methylpyridin-2-yl)-6-(pyridin-2-ylmethoxy)-1H-

pyrazolo[3,4-b]pyrazine (13). The title compound was prepared using

method B (library format, resynthesis) with 2-bromo-3-methylpyr-

idine. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (m, 1H), 8.38 (s, 1H), 7.74

(td, J = 7.8, 1.7 Hz, 1H), 7.64 (dd, J = 7.8, 1.6 Hz, 1H), 7.52 (d, J = 7.8

Hz, 1H), 7.25 (m, 2H), 5.85 (s, 2H), 4.09 (s, 3H), 2.55 (s, 3H). MS

(ES⁺): m/z 333.2 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

1-Methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-

pyrazolo[3,4-b]pyrazine (14). The title compound was prepared using

1

81% yield. H NMR (300 MHz, DMSO-d₆) δ 8.72 (s, 1H), 4.06 (s,

3H). MS (ES⁺): m/z 295.0 (M + 1).

Step 7: synthesis of 3-iodo-1-methyl-6-(pyridin-2-ylmethoxy)-1H-

pyrazolo[3,4-b]pyrazine (42). A solution of potassium tert-butoxide

(8.38 g, 74.7 mmol) in THF (170 mL) was added dropwise over 20

min to a mixture of 6-chloro-3-iodo-1-methyl-1H-pyrazolo[3,4-b]-

pyrazine (41) (20 g, 68 mmol) and pyridin-2-ylmethanol (7.22 mL,

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Journal of Medicinal Chemistry

Article

method A (Negishi coupling) with 3-bromo-4-methylpyridine. ¹H

NMR (400 MHz, DMSO-d₆) δ 9.00 (s, 1H), 8.60 (br d, J = 4.8 Hz,

1H), 8.49 (d, J = 5.0 Hz, 1H), 8.43 (s, 1H), 7.86 (dd, J = 7.6, 7.6 Hz,

1H), 7.63 (d, J = 7.8 Hz, 1H), 7.35−7.43 (m, 2H), 5.61 (s, 2H), 4.04

(s, 3H), 2.53 (s, 3H). MS (ES⁺): m/z 333.1 (M + 1). UHPLC: 100%

(UV), 100% (ELSD).

Step 2: synthesis of (3,5-dichloropyrazin-2-yl)(4-methylpyridin-3-

yl)methanone (45). A solution of 2,2,6,6-tetramethylpiperidine (30

mL, 180 mmol) in THF (1.5 L) was cooled to −40 °C. n-Butyl lithium

(2.5 M solution, 80 mL, 200 mmol) was slowly added, and the

reaction mixture was allowed to warm to 0 °C and stir for 20 min. The

reaction mixture was cooled to −80 °C, and a solution of 2,6-

dichloropyrazine (25 g, 170 mmol) in tetrahydrofuran (200 mL) was

added dropwise. After the reaction mixture had stirred for 1 h at −80

°C, ethyl 4-methylpyridine-3-carboxylate (44) (23 g, 140 mmol) was

slowly added, and the resulting mixture was stirred at −80 °C for 2 h.

The reaction was quenched by slow addition of acetic acid (15 mL),

after which it was allowed to warm to rt. The mixture was then diluted

with ethyl acetate (1.2 L) and washed sequentially with aqueous

hydrochloric acid (0.1 M, 2 × 500 mL), saturated aqueous sodium

bicarbonate solution (500 mL), and saturated aqueous sodium

chloride solution (500 mL). The organic layer was ﬁltered and

concentrated in vacuo. Twenty batches following this protocol were

1-Methyl-6-(pyridin-2-ylmethoxy)-3-(4-(triﬂuoromethyl)pyridin-

3-yl)-1H-pyrazolo[3,4-b]pyrazine (15). The monofumarate salt of the

title compound was prepared using method A (Negishi coupling) with

3-bromo-4-(triﬂuoromethyl)pyridine followed by treatment with a

solution of fumaric acid in a minimal volume of methanol and

1

recrystallization. H NMR (400 MHz, CD₃OD) δ 9.05 (s, 1H), 8.88

(d, J = 5.3 Hz, 1H), 8.58 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H), 8.33 (s, 1H),

7.87−7.94 (m, 2H), 7.69 (br d, J = 7.8 Hz, 1H), 7.39−7.43 (m, 1H),

6.75 (s, 2H), 5.66 (s, 2H), 4.09 (s, 3H). MS (ES⁺): m/z 387.1 (M +

1). Anal. Calcd for C₁₈H₁₄F₃N₆O: C, 52.59; H, 3.41; N, 16.73; F,

11.34. Found: C, 52.55; H, 3.10; N, 16.56; F, 11.30.

3-(3-(Diﬂuoromethoxy)pyridin-2-yl)-1-methyl-6-(pyridin-2-ylme-

thoxy)-1H-pyrazolo[3,4-b]pyrazine (16). The title compound was

prepared using method A (Negishi coupling) with 2-bromo-3-

1

combined to give 620 g of 45 as a brown oil in 82% yield. H NMR

(400 MHz, CDCl₃) δ 8.63 (d, J = 5.0 Hz, 1H), 8.58 (s, 1H), 8.53 (s,

1H), 7.32 (d, J = 5.0 Hz, 1H), 2.63 (s, 3H). MS (ES⁺): m/z 268.1 (M

+ 1).

1

(diﬂuoromethoxy)pyridine. H NMR (400 MHz, CDCl₃) δ 8.74 (br

d, J = 4.6 Hz, 1H), 8.66 (br d, J = 4.5 Hz, 1H), 8.41 (s, 1H), 7.78 (br

dd, J = 7.7, 7.6 Hz, 1H), 7.73 (br d, J = 8.2 Hz, 1H), 7.55 (br d, J = 7.8

Hz, 1H), 7.40 (dd, J = 8.3, 4.8 Hz, 1H), 7.30 (br dd, J = 7.1, 5.1 Hz,

1H), 6.74 (t, J_HF= 74.7 Hz, 1H), 5.67 (s, 2H), 4.12 (s, 3H). MS

(ES⁺): m/z 385.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

3-(5-Chloro-4-methylpyridin-3-yl)-1-methyl-6-(pyridin-2-ylme-

thoxy)-1H-pyrazolo[3,4-b]pyrazine (17). The title compound was

prepared using method A (Negishi coupling) with 3-bromo-5-chloro-

Step 3: synthesis of 6-chloro-1-ethyl-3-(4-methylpyridin-3-yl)-1H-

pyrazolo[3,4-b]pyrazine (46a). Triethylamine (99%, 0.66 mL, 4.7

mmol) was added to a solution of (3,5-dichloropyrazin-2-yl)(4-

methylpyridin-3-yl)methanone (45) (208 mg, 0.776 mmol) in toluene

(4 mL). After addition of the oxalate salt of ethyl hydrazine (350 mg,

2.33 mmol), the reaction mixture was stirred at rt for 72 h. Water and

ethyl acetate were added, and the mixture was ﬁltered through Celite.

The aqueous layer was extracted twice with ethyl acetate, and the

combined organic layers were dried over MgSO₄, ﬁltered, and

concentrated in vacuo. Puriﬁcation via silica gel chromatography

(gradient: 0−100% ethyl acetate in heptane) aﬀorded 65 mg of 46 as a

1

4-methylpyridine. H NMR (400 MHz, CDCl₃) δ 8.87 (s, 1H), 8.68

(br d, J = 4.9 Hz, 1H), 8.60 (s, 1H), 8.35 (s, 1H), 7.82 (ddd, J = 7.8,

7.6, 1.6 Hz, 1H), 7.59 (br d, J = 7.8 Hz, 1H), 7.34 (br dd, J = 7.3, 5.2

Hz, 1H), 5.71 (s, 2H), 4.12 (s, 3H), 2.60 (s, 3H). MS (ES⁺): m/z

367.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

1

1-Methyl-6-(pyridin-2-ylmethoxy)-3-(4-methyl-pyridazin-3-yl)-

1H-pyrazolo[3,4-b]pyrazine (18). The title compound was prepared

using method A (singleton format) with 3-bromo-4-methyl-pyridazine

yellow solid in 32% yield. H NMR (400 MHz, CDCl3) δ 9.20 (br s,

1H), 8.59 (s, 1H), 8.55 (br d, J = 5.2 Hz, 1H), 7.34−7.37 (m, 1H),

4.62 (q, J = 7.3 Hz, 2H), 2.64 (br s, 3H), 1.63 (t, J = 7.3 Hz, 3H). MS

(ES⁺): m/z 274.0 (M + 1).

Step 4: synthesis of 1-ethyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-

ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (20). The title compound

was prepared according to step 7 of the preparation of advanced

intermediate 42 using 6-chloro-1-ethyl-3-(4-methylpyridin-3-yl)-1H-

pyrazolo[3,4-b]pyrazine (46a) and pyridin-2-ylmethanol as reactants.

¹H NMR (400 MHz, CDCl3) δ 9.08 (br s, 1H), 8.66 (ddd, J = 4.9, 1.8,

0.9 Hz, 1H), 8.51 (d, J = 5.0 Hz, 1H), 8.35 (s, 1H), 7.77 (ddd, J = 7.8,

7.6, 1.8 Hz, 1H), 7.53−7.57 (m, 1H), 7.26−7.31 (m, 2H), 5.66 (s,

2H), 4.50 (q, J = 7.2 Hz, 2H), 2.59 (s, 3H), 1.56 (t, J = 7.3 Hz, 3H).

MS (ES⁺): m/z 347.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

Preparation of 1-Cyclopropyl-3-(4-methylpyridin-3-yl)-6-(pyridin-

2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (21). Step 1: synthesis of

6-chloro-1-cyclopropyl-3-(4-methylpyridin-3-yl)-1H-pyrazolo[3,4-b]-

pyrazine (46b). The title compound was prepared according to step 3

of the preparation of compound 20 using the hydrochloride salt of

cyclopropylhydrazine as reactant. ¹H NMR (400 MHz, CDCl3) δ 9.16

(br s, 1H), 8.59 (s, 1H), 8.54 (d, J = 5.2 Hz, 1H), 7.32 (br d, J = 5.2

Hz, 1H), 3.97−4.03 (m, 1H), 2.61 (br s, 3H), 1.40−1.46 (m, 2H),

1.22−1.28 (m, 2H). MS (ES⁺): m/z 286.0 (M + 1).

1

in 18% yield. H NMR (400 MHz, CDCl₃) δ 9.02 (br d, J = 5.1 Hz,

1H), 8.60 (ddd, J = 4.6, 1.5, 1.0 Hz, 1H), 8.35 (s, 1H), 7.69 (td, J =

7.6, 1.8 Hz, 1H), 7.47 (br d, J = 7.8 Hz, 1H), 7.35 (dd, J = 5.3, 1.0 Hz,

1H), 7.21 (m, 1H), 5.60 (s, 2H), 4.07 (s, 3H), 2.51 (s, 3H). MS (ES⁺):

m/z 334.1 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

3-(1,5-Dimethyl-1H-pyrazol-4-yl)-1-methyl-6-(pyridin-2-ylme-

thoxy)-1H-pyrazolo[3,4-b]pyrazine (19). The title compound was

prepared using method A (singleton format) with 4-bromo-1,5-

1

dimethyl-1H-pyrazole in 51% yield. H NMR (400 MHz, CDCl₃) δ

8.66 (ddd, J = 4.9, 1.6, 0.8 Hz, 1H), 8.29 (s, 1H), 8.22 (br s, 1H), 7.77

(ddd, J = 7.8, 7.6, 1.8 Hz, 1H), 7.55 (br d, J = 7.8 Hz, 1H), 7.27−7.31

(m, 1H), 5.65 (s, 2H), 4.02 (s, 3H), 3.88 (s, 3H), 2.65 (s, 3H). MS

(ES⁺): m/z 336.0 (M + 1). UHPLC: 100% (UV), 100% (ELSD).

Preparation of 1-Ethyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-yl-

methoxy)-1H-pyrazolo[3,4-b]pyrazine (20). Step 1: synthesis of

ethyl 4-methylpyridine-3-carboxylate (44). A mixture of 3-bromo-4-

methylpyridine (43) (160 g, 0.930 mol), 1,1′-bis(diphenylphosphino)-

ferrocene (103 g, 0.186 mol), triethylamine (257 mL, 1.84 mol), and

palladium(II) acetate (20.8 g, 92.7 mmol) in ethanol (2.4 L) and DMF

(1.2 L) was placed in a sealed autoclave and stirred under a carbon

monoxide atmosphere (2.5 MPa) at 100 °C for 18 h. The mixture was

cooled to rt and concentrated in vacuo to remove ethanol. Solids were

removed via ﬁltration, and the ﬁlter cake was washed with ethyl acetate

(1 L). The combined ﬁltrates were diluted with additional ethyl acetate

(1 L) and washed with a saturated aqueous sodium chloride solution

(3 × 1 L). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude products from four runs of this

reaction were combined and puriﬁed by chromatography on silica gel

(gradient: 4−9% ethyl acetate in petroleum ether), aﬀording 430 g of

44 as a colorless oil in 70% yield. ¹H NMR (400 MHz, CDCl3) δ 9.05

(s, 1H), 8.53 (d, J = 5.0 Hz, 1H), 7.15 (d, J = 5.0 Hz, 1H), 4.38 (q, J =

7.1 Hz, 2H), 2.60 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). GCMS (ES⁺): m/z

166.2 (M + 1).

Step 2: synthesis of 1-cyclopropyl-3-(4-methylpyridin-3-yl)-6-

(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (21). The title

compound was prepared according to step 7 of the preparation of

advanced intermediate 42 using 6-chloro-1-cyclopropyl-3-(4-methyl-

pyridin-3-yl)-1H-pyrazolo[3,4-b]pyrazine (46b) and pyridin-2-ylme-

1

thanol as reactants. H NMR (400 MHz, CDCl₃) δ 9.07 (br s, 1H),

8.67 (ddd, J = 4.9, 1.8, 0.8 Hz, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.35 (s,

1H), 7.77 (ddd, J = 7.8, 7.6, 1.8 Hz, 1H), 7.55−7.59 (m, 1H), 7.26−

7.31 (m, 2H), 5.68 (s, 2H), 3.83−3.89 (m, 1H), 2.57 (s, 3H), 1.33−

1.38 (m, 2H), 1.13−1.19 (m, 2H). MS (ES⁺): m/z 359.1 (M + 1).

UHPLC: 100% (UV), 100% (ELSD).

875

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Journal of Medicinal Chemistry

Article

(7) The structure of ADX48621 (Dipraglurant) was ﬁrst disclosed in

international nonproprietary names for pharmaceutical substances

(INN). WHO Drug Information; World Health Organization: Geneva,

Switzerland, 2009; Vol. 23, p 328.

(8) (a) Morin, N.; Gregoire, L.; Gomez-Mancilla, B.; Gasparini, F.;

́

Di Paolo, T. Effect of the metabotropic glutamate receptor type 5

ASSOCIATED CONTENT

* Supporting Information

NHP PD-LID model protocol and expanded SAR tables with

mGluR5 binding and functional activity ranges. This material is

available free of charge via the Internet at http://pubs.acs.org.

PF470 (14), also known as PF-06297470, is now commercially

available at Sigma-Aldrich (product no. PZ0219).

■

S

antagonists MPEP and MTEP in parkinsonian monkeys. Neuro-

pharmacology 2010, 58, 981−986. (b) Gregoire, L.; Morin, N.;

́

Ouattara, B.; Gasparini, F.; Bilbe, G.; Johns, D.; Vranesic, I.;

Sahasranaman, S.; Gomez-Mancilla, B.; Di Paolo, T. The acute

antiparkinsonian and antidyskinetic effect of AFQ056, a novel

metabotropic glutamate receptor type 5 antagonist, in L-DOPA-

treated parkinsonian monkeys. Parkinsonism Related Disord. 2011, 17,

270−276. (c) Addex’s ADX48621 effective in preclinical Parkinson’s

disease studies; Addex Pharmaceuticals Press Release: Geneva,

Switzerland, 2010; http://www.addextherapeutics.com/investors/

press-releases/.

AUTHOR INFORMATION

Corresponding Author

*Phone: +1 617 395 0640. Fax: +1 860 686 5285. E-mail: Lei.

Zhang3@p ﬁ zer.com.

■

Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

We thank MOTAC Neuroscience (Beijing, People’s Republic

of China) for performing the NHP PD-LID study. We also

thank the Pﬁzer ADME technology group for generating the in

vitro pharmacokinetic data in support of the SAR eﬀort.

ABBREVIATIONS USED

■

2T, two-tissue; CL_int, intrinsic clearance; F, oral bioavailability;

FPEB, 3-ﬂuoro-5-[(pyridin-3-yl)ethynyl]benzonitrile; HLM,

human liver microsomes; LipE, lipophilicity eﬃciency; MPEP,

2-methyl-6-(phenylethynyl)pyridine; MPEPy, 3-methoxy-5-

pyridin-2-ylethynylpyridine; MPTP, 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine; MPO, multiparameter optimization;

MTEP, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine; NHP, non-

human primate; PDE, phosphodiesterase; PD-LID, ^L-DOPA-

induced dyskinesia in Parkinson’s disease; PET, positron

emission tomography; RO, receptor occupancy; RLM, rat

liver microsomes; RRCK, RR canine kidney; TAC, time-activity

curve; TI, therapeutic index; V_ss, steady-state volume of

distribution; V_t, volume of distribution

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877

dx.doi.org/10.1021/jm401622k | J. Med. Chem. 2014, 57, 861−877

Article Doi

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin- 3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): A highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator

DOI: 10.1021/jm401622k

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