Structure-Based Optimization of Naphthyridones into Potent ATAD2 Bromodomain Inhibitors

Article

pubs.acs.org/jmc

Structure-Based Optimization of Naphthyridones into Potent ATAD2

Bromodomain Inhibitors

Paul Bamborough,*^,†Chun-wa Chung,^†Rebecca C. Furze,^‡Paola Grandi,^∥Anne-Marie Michon,^∥

Robert J. Sheppard,^‡,⊥Heather Barnett,^§Hawa Diallo,^‡David P. Dixon,^†Clement Douault,^‡

Emma J. Jones,^†Bhumika Karamshi,^†Darren J. Mitchell,^‡Rab K. Prinjha,^‡Christina Rau,^∥

Robert J. Watson,^‡Thilo Werner,^∥and Emmanuel H. Demont*^,‡

‡

§

^†Molecular Discovery Research, Epinova Discovery Performance Unit, and Flexible Discovery Unit, GlaxoSmithKline Medicines

Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

^∥Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany

S

* Supporting Information

ABSTRACT: ATAD2 is a bromodomain-containing protein

whose overexpression is linked to poor outcomes in a number

of diﬀerent cancer types. To date, no potent and selective

inhibitors of the bromodomain have been reported. This

article describes the structure-based optimization of a series of

naphthyridones from micromolar leads with no selectivity over

the BET bromodomains to inhibitors with sub-100 nM

ATAD2 potency and 100-fold BET selectivity.

INTRODUCTION

■

The bromodomain-containing protein ATAD2 (ATPase family,

AAA domain containing 2), also known as ANCCA (AAA

nuclear coregulator cancer-associated protein), is a promising

oncology target. Increased expression correlates with poor

outcome in a range of cancers, and ATAD2 knockdown has

been shown to modulate multiple relevant tumor cell growth

factors.¹⁻³The fact that ATAD2 contains a bromodomain

makes it an attractive area for research, as similar modules are

known to be tractable to drug discovery in the tandem-

bromodomain BET (bromodomain and extra terminal) family

proteins (BRD2,3,4 and T).^4,5Despite this, relatively little work

has been published, and there is a great need for small

molecules with which to investigate phenotypic responses to

inhibition of the ATAD2 bromodomain. Until very recently,

the most potent inhibitors of the ATAD2 bromodomain were

weak (≥200 μM) fragments, which are not suitable for use as

cellular chemical probes and for which no optimization has

been reported.

In our preceding article, we described the discovery of the

ﬁrst reported low-micromolar inhibitors of the ATAD2

bromodomain (Figure 1).⁶A weak hit 1 (Figure 1) was

synthesized within an ATAD2-targeted portion of an array

based upon a quinolinone template. The crystallographic

binding mode to the bromodomain of ATAD2 of analogues

such as 2 was characterized, conﬁrming that the napthyridone

acts as an acetyl-lysine (KAc) mimetic via direct and through-

Figure 1. From hit to micromolar ATAD2 inhibitor. pIC₅₀s of

compounds 1−3 in ATAD2 peptide- and ligand-based TR-FRET

assays and against BRD4 BD1.

water hydrogen-bonded interactions to Asn1064 and Tyr1021

(Figure 2b). The basic piperidine substituent makes an

important additional contribution to ATAD2 binding through

its interaction with Asp1071. Exploration of SAR at the C5

position of the naphthyridone demonstrated that 3-pyridyl

derivatives provided a further boost to ATAD2 activity and

resulted in the micromolar ATAD2 lead 3.

Because of the extensive eﬀects of BET inhibitors,⁷high

selectivity over these proteins is required in chemical probes of

other bromodomains. In a cellular probe for ATAD2, we aimed

to achieve at least 100-fold selectivity over the BET family.

Received: May 21, 2015

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Attempts to gain this selectivity through further exploration of

the C5-position met with limited success, with most analogues

being more active against the BET family, exempliﬁed by the N-

terminal bromodomain of BRD4 (BRD4 BD1), than ATAD2.

We now continue the story of our attempts to increase ATAD2

potency and introduce selectivity over the BET bromodomains.

of the piperidine seemed to oﬀer a suitable vector (Figure 2b).

We ﬁrst tried to interact with the lipophilic part of the RVF

RESULTS AND DISCUSSION

■

As this work progressed, we moved away from the peptide-

based assay we had used to develop compound 3 to an

alternative TR-FRET competition binding assay, in which a

ﬂuorescently labeled naphthyridone analogue of 3 was used

instead of a peptide. Data generated for compounds in both

assays showed that the two were in good agreement (r²= 0.94

for 386 compounds over the TR-FRET IC₅₀range 100 μM to

100 nM; see Figure S1a, Supporting Information). ATAD2

pIC₅₀data reported here were generated using the ligand-based

TR-FRET assay. For completeness, data from the peptide-

based assay is also shown in Table S1, Supporting Information.

Throughout the optimization, we also conﬁrmed that the

activity against the recombinant bromodomain was retained

against ATAD2 protein from a more physiological source. The

ATAD2 Bromosphere assay measures competitive binding to

endogenous full-length ATAD2 from cell lysate. The

correlation between the ATAD2 Bromosphere assay and the

recombinant TR-FRET assay was also good (r²> 0.67 for 237

compounds over a range of TR-FRET IC₅₀between 100 μM

and 100 nM; Figure S1b, Supporting Information).

Exploration of the 3′ Position to Find Lipophilic

Interactions with the RVF Shelf. Having found diﬃculty in

improving on the 3-pyridyl substituent at the naphthyridone C5

position of 3, we considered ways to interact with other parts of

the ATAD2 binding site. For the BET-family bromodomains,

some areas outside the immediate KAc site have proved to be

valuable in optimizing potency. One such area is the WPF shelf,

named after the conserved Trp−Pro−Phe motif located nearby

(Figure 2a).⁸In the BRD4 N-terminal bromodomain (BRD4

BD1), key residues of the WPF shelf include Trp81 and Pro82

of the WPF motif, Met149, and especially Ile146. Potent BET

inhibitors typically exploit the WPF shelf using aromatic or

lipophilic substituents. In one recent example, aniline

substituents projecting from a tetrahydroquinoline core onto

the BET WPF shelf gained signiﬁcant extra potency, and a

similarly positioned phenyl ring is an essential part of ﬁrst-

generation BET inhibitors I-BET762 (Figure 2a) and JQ-1.⁹⁻¹¹

A similar shallow shelf exists in ATAD2, but the amino acids

surrounding it diﬀer considerably from the BET bromodo-

mains. As the BET WPF motif is replaced by RVF (Arg1007−

Val1008−Phe1009) in ATAD2, this region has been called the

RVF shelf.¹²Another important diﬀerence is the presence of

Arg1077 in ATAD2 instead of Met149 (BRD4 numbering)

(Figure 2b and Figure S2, Supporting Information). Together,

these changes result in a more polar and ﬂexible environment

on the RVF shelf in ATAD2 that is more challenging to interact

with productively than in BRD4. While the rims of the two

bromodomain shelves diﬀer considerably, the ﬂoors are more

similar. In ATAD2, the ﬂoor is formed by gatekeeper residue

Ile1074 (Figure 2b), which is identical to the corresponding

residue in the second BET bromodomains, and only

conservatively diﬀers from the ﬁrst (Ile146 and Val439 in

BRD4 BD1 and BD2, respectively).

Figure 2. (A) Acetyl-lysine binding site of BRD4 BD1 bound to

literature inhibitor I-BET762 (PDB code 3p5o)¹⁰showing detailed

interactions (left) and electrostatic charge colored protein surface

(right). (B) X-ray structure of ATAD2 bound to 2 (PDB code 5a5q),

in the same orientation. Hydrogen bonds are shown as yellow dashed

lines.

shelf surface produced by Ile1074, which is the closest part of

the shelf to the piperidine ring of 3. Modeling suggested that an

ether linker could direct lipophilic groups toward this position

in a low-energy conformation.

This hypothesis could be explored rapidly in the C5-H

naphthyridone template, via O-alkylation of the trans azido

protected hydroxy-piperidine 5, which was easily accessible

from the known epoxide 4 (Scheme 1).¹³Reduction of the

azide generated protected 3-alkoxy 4-amino piperidines 6

suitable for palladium-catalyzed coupling with the chloro

naphthyridines 7a,b, as previously described.⁶Deprotection

then followed under acidic conditions. As well as giving access

to racemic products, this route also enabled the isolation of

inhibitors as single enantiomers, since the enantiomers of the

azides, BOC-protected naphthyridines, or the ﬁnal compounds

could be easily separated by chiral chromatography.

The SAR (Table 1) shows a size- and lipophilicity-dependent

increase in potency up to a point, with acyclic groups producing

relatively ineﬀective ATAD2 inhibitors (9−12). Alpha branch-

ing (as in 10) was especially poorly tolerated compared to

linear or β-branched alkyl chains (e.g., 11 and 12). The

cyclohexyl derivative 13 appeared to be the optimal cycloalkyl

group, being marginally more ligand-eﬃcient than the cyclo-

pentyl (14) and comparable to the cycloheptyl (15). After

separation of the two enantiomers of compound 13, one

enantiomer (17) was signiﬁcantly more active than the other

(16) by a factor of 1.3 logs. We judged that although the

increase in potency in moving from 2 to 17 was achieved at the

expense of some ligand eﬃciency and was driven by

lipophilicity, this was acceptable to counter the hydrophilicity

We inspected the ATAD2 crystal structure complex with 3 to

ﬁnd ways to target the RVF shelf. The C3′ equatorial position

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of the naphthyridone core, producing more balanced molecules

with greater logD to promote cell permeability.

Scheme 1. Synthesis of Key Trans Piperidines

A 2.0 Å crystal structure of ATAD2 bound to compound 13

was rapidly solved (Figure 3a). The addition of the 3′

substituent does not change the overall binding mode of the

naphthyridone, which is unmoved relative to the bound

structure of 2. Interestingly, only the single (R,R) isomer of

the trans racemic compound 13 was seen to bind in the

ATAD2 active site (Figure S3, Supporting Information). The

structure conﬁrmed our intended binding mode, with the 3′-

substituent cyclohexyl group positioned on the RVF shelf, in

direct hydrophobic contact with Ile1074. The addition of the

cyclohexyl substituent displaces two water molecules from

ATAD2 that were visible in the complex with 2. Both appear to

be weakly bound. For example, one displaced water molecule,

labeled H₂O_(R1077)in Figure 3a, forms only a single visible

hydrogen bond to the Arg1077 guanidine group and is

otherwise surrounded by the aliphatic side chains of Val1008,

Leu1073, and Ile1074.

a

Reagents and conditions: (a) NaN₃, DMF/acetone/water, 80°C,

63%; (b) NaH, DMF, 0°C then RI or ROTf, 0 °C to room

temperature, 10−100%; (c) Pd/C, MeOH, H₂(1 bar), 69−100% or

PPh₃, THF, room temperature then water, reﬂux, 69−79%; (d) for R₂

= H: LiHMDS, Caddick catalyst, THF, 80−120 °C, microwave, 13−

95%; or for R₂= Bn: BrettPhos, BrettPhos precatalyst, THF, tBuONa,

room temperature, 65−95%; (e) for R₂= H: TFA, CH₂Cl₂, room

temperature, 73−92%; (f) for R₂= Bn: H₂, Pd/C, MeOH, room

temperature, 99% then TFA, CH₂Cl₂, room temperature, 84%.

For comparison, the BRD4 BD1 X-ray crystal structure with

compound 13 was obtained by cocrystallization (Figure 3b).

The naphthyridone of 13 binds to the acetyl-lysine pocket of

BRD4 in a similar way to that of 3.⁶As with ATAD2, the

a

Table 1. Data for C3′-Substituents in the C5-H Naphthyridone Series

a

Ligand eﬃciency (LE) ≈ 1.37 × pIC₅₀/(number of heavy atoms). For statistics, see Table S1, Supporting Information.

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Figure 3. (A) Crystal structure of ATAD2 bound to 13 (green, PDB code 5a81), superimposed on that of 2 (orange, PDB code 5a5q) using alpha-

carbon atoms of the active sites. The water molecule labeled H₂O_(R1077)on the RVF shelf close to Arg1077 is present in the complex with 2 and is

displaced by 13. (B) Crystal structure of BRD4 BD1 bound to 13 (green, PDB code 5a85). Dashed yellow lines indicate hydrogen bonds.

electron density in the BRD4 BD1 X-ray structure was

consistent with the (R,R) enantiomer. The cyclohexyl ring

binds on the BRD4 BD1 WPF shelf, making lipophilic contact

with the Ile146 side chain. As this hydrophobic interaction

(with Ile1074 and Ile146 in ATAD2 and BRD4 BD1,

respectively) is similar in both bromodomains, it is not

surprising that the BRD4 BD1 potency tracks the ATAD2

potency closely for these and similar compounds (Table 1).

From the mixture of the two trans enantiomers of 13, the

(R,R) enantiomer was found to bind to both ATAD2 and

BRD4 crystallographically. We assumed that this was the more

potent of the two separated enantiomers and that, therefore,

the more potent of the two separated enantiomers, 17, had

(R,R) stereochemistry. Later synthesis of enantiomerically pure

material via chiral separation of azide intermediate 5 (Scheme

1)¹⁴followed by crystallographic binding mode determination

of the most active enantiomer showed that, indeed, the most

potent single enantiomer was always the one associated with

(R,R) stereochemistry (data not shown). We checked periodi-

cally to ensure that this remained true. From hereon, it is

assumed that the (R,R) enantiomer is systematically more

potent against ATAD2 than (S,S).

the improvements in potency from modiﬁcation at the C3′

position could be boosted further if other changes were

additive. In our previous article, we described SAR at the

naphthyridone C5-position.⁶We introduced some of our

favored C5-substituents, including the 3-pyridyl group of 3,

into C3′-derivatized molecules according to Scheme 2. The

derivatives 8 were selectively brominated at C5 to give 18,

which could be coupled with the required boronic acids to give

19. Acidic deprotection of these intermediates gave the

corresponding inhibitors in moderate to good yields.

Data for two example C5, C3′ disubstituted derivatives are

presented in Table 2. As reported previously for the 3′-H

piperidine analogues, the addition of 3-pyridyl substituents at

the C-5 position improved ATAD2 potency signiﬁcantly,

conserving ligand eﬃciency (compare 20 and 22 to 17).

Consistent with the data shown in Table 1 for 5-H

naphthyridones, in the C5-(3-pyridyl) analogues the (R,R)

enantiomer 20 was 1.3 logs more potent than the (S,S)

enantiomer 21. For the ﬁrst time, these compounds clearly

demonstrated the possibility of achieving our target ATAD2

activity (pIC₅₀>7.0). In the case of compound 22, this

milestone was conﬁrmed by potent activity measured against

endogenous ATAD2 in the chemoproteomic Bromosphere assay

(Table 2).

Combining C5 and Lipophilic C3′ Substituents. Seeing

little diﬀerence in the position of the naphthyridone between

the ATAD2-bound X-ray structures of 2 and 13, we hoped that

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or water-bridged interactions with Arg1077. If direct or indirect

H-bonds could not be formed, then we hoped at least that

electronegative groups would be more tolerated in ATAD2

because of the more positive electrostatic surface in this area

while being detrimental to BET activity by virtue of its greater

lipophilicity.

Scheme 2

Polar C3′ Substituents. As before, derivatives were ﬁrst

synthesized in the naphthyridone C5-H template. In most

cases, these were prepared from the racemic trans hydroxy

azido piperidine 5 (Scheme 1), and the enantiomers were

separated for selected examples. In some cases (examples 26,

31−33, 44), the alkylation with the C3′ substituent was

performed after the Buchwald coupling between 7b and the

protected amine 23, which was derived from the hydroxyl

derivative 5 (Scheme 3). Deprotection of the secondary alcohol

gave 24, which, after alkylation and further deprotection, gave

the desired inhibitors.

a

Scheme 3

a

Reagents and conditions: (a) NBS, CHCl₃, −10 °C, 84−95%; (b)

Pd(OAc)₂, K₂CO₃, cataCXium A, ArB(OH)₂, 1,4-dioxane/water, 100

°C, microwave, 46−87%; (c), TFA, CH₂Cl₂, room temperature (R₁=

H, 26−90%) or TFA reﬂux (R₁= Bn, 39−99%).

Also for the ﬁrst time, the combination of C5 and C3′ groups

in compounds 20 or 22 gave a modest but signiﬁcant window

of selectivity for ATAD2 over BRD4 (≥0.7 log diﬀerence

between the two pIC₅₀values in TR-FRET assays). While

encouraging, we felt that this level of selectivity was still too

small to be able to distinguish any phenotypic readout due to

ATAD2 from residual BET activity. We therefore looked for

ways to widen the selectivity window. Up to this point, the

selection of substituents at the 3′ position had been quite

limited and did not seek to exploit the diﬀerences between the

ATAD2 RVF shelf and the BET WPF shelf outlined above. The

ATAD2 environment is signiﬁcantly more polar, due to the

presence of Arg1007 and Arg1077, than in BRD4 (Trp81 and

Met149). Figure 3 shows the diﬀerent surroundings of the 4-

position of the 3′-cyclohexyl moiety of 13 when bound to

ATAD2 and BRD4. As mentioned above, the loss of a water

molecule H₂O_(R1077)bound to the Arg1077 side chain had been

seen upon the addition of the cyclohexyl C3′ substituent. We

speculated that introducing hydrogen-bond acceptor groups in

this region might improve ATAD2 activity either through direct

a

Reagents and conditions: (a) TBDMSCl, imidazole, CH₂Cl₂, room

temperature, 71%; (b) H₂, Pd/C, EtOH, room temperature, 95%; (c)

BrettPhos, BrettPhos precatalyst, tBuONa, THF, room temperature to

60 °C, 97%; (d) TBAF, THF, room temperature, 71%; (e) chiral

separation of enantiomers; (f) ROTf, tBuOK, THF, 0 °C to room

temperature, 10−65% or NaH, THF (or DMF), 0 °C then ROTf, 0

°C to room temperature, 37−65%; (g) TFA, 60 °C to reﬂux, 45−94%.

Examples of such compounds with increased polarity in their

C3′ substituents are shown in Table 3. Most of the racemic

mixtures (e.g., 25−27) had ATAD2 activity comparable to that

of the racemic cyclohexyl benchmark 13 (pIC₅₀5.5). As before,

when the mixtures were separated, most of the activity resided

in a single enantiomer (compare 29 to 28). The most

a

Table 2. Eﬀect of C5-(3-Pyridine) Substituents

Compound

ATAD2 pIC₅₀

ATAD2 LE

ATAD2 Bromosphere pIC₅₀

BRD4 BD1 pIC₅₀

Δ

Solubility μM

LogD

17

20

21

22

5.6

6.7

5.4

6.9

0.28

0.27

0.22

0.28

5.4

6.5

5.4

5.8

4.9

6.2

0.2

0.9

0.5

0.7

220

170

196

494

3.3

4.0

4.2

2.2

7.4

a

Δ = diﬀerence between ATAD2 and BRD4 BD1 FRET pIC₅₀. For statistics, see Table S1, Supporting Information.

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Table 3. Polar C3′ Substituents

a

Δ = diﬀerence between ATAD2 and BRD4 BD1 FRET pIC₅₀. For statistics, see Table S1, Supporting Information

promising polar analogue was the cyclic sulfone 30. When

chirally separated, the more active enantiomer 32 showed

improved ATAD2 activity over the chiral cyclohexyl 17. More

importantly, consistent with our strategy for increasing

selectivity over BRD4 by increasing negative polarity, both

the tetrahydropyran 29 and cyclic sulfone 32 showed lower

BRD4 activity than 17. Further analogues using similar ring

systems (e.g., 33) did not show any advantage, as was also the

case when extending the linker between the piperidine and the

substituent (e.g., 34).

To better understand the interactions made by the cyclic

sulfone and to conﬁrm the identity of the more active

enantiomer, a soak of the racemic cyclic sulfone 30 into

ATAD2 crystals led to the X-ray structure shown in Figure 4.

The binding mode is very similar to that of 13. Again, the (R,R)

isomer can be seen unambiguously binding within the active

site (Figure S3, Supporting Information). The main diﬀerences

from the structure of 13 are the polar interactions made by the

sulfone oxygen atoms, each of which accepts hydrogen bonds

from the guanidinium group of Arg1077. The ﬁrst sulfone

oxygen lies in the position occupied by H₂O_(R1077), the

Arg1077-bound water molecule in the complex with 2 (Figure

4c). The second sulfone oxygen also hydrogen bonds to the

Arg1077 terminal NH₂group and is able to make a second

interaction with the terminal NH₂group of Arg1007, which

moves slightly toward the sulfone.

The enhanced selectivity over BRD4 of 32 relative to 17 is

due to both increased ATAD2 potency and decreased BET

activity (Table 3). The ATAD2 potency gain arises from the

new direct hydrogen bonds to the arginines and the

displacement of the weakly bound water molecules. The

reduction of BET activity presumably results from placing polar

sulfone oxygen atoms in an unfavorable lipophilic location near

Trp81 and Met149 (Figure 3b). The tetrahydropyran 29 is

intermediate in polarity between the cyclohexyl 17 and cyclic

sulfone 32 and shows intermediate selectivity.

Combining C5 and Polar C3′ Substituents. The

tetrahydropyran and the cyclic sulfone C3′ substituents oﬀered

the best compromise of structural complexity, ATAD2 activity,

and BET selectivity. Next, we assessed the impact of

reintroduction of C5-(3-pyridine) groups on the overall proﬁle

of our fully functionalized inhibitors, prepared according to

Scheme 2. In agreement with earlier results, C5-(3-pyridyl)

substituents were about 1 log more potent against ATAD2 than

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in potency than the cyclic sulfones by ∼0.3 logs (compare 35 to

36, 37 to 38, and 39 to 40).

Unexpectedly, addition of C5-(3-pyridine) substituents to

compounds with polar C3′ groups also improved the selectivity

for ATAD2 over BRD4 BD1 (compare, e.g., 38 to 32). 38 was

the ﬁrst compound whose activity in the ATAD2 FRET assay

was over 2 logs greater than in BRD4 BD1. Interestingly,

introduction of the (3,5)-pyrimidine at C5 gave somewhat

improved BET selectivity (in the case of the tetrahydropyran

41 compared to 35, 37, or 39), but this came at the expense of

some ATAD2 potency. This is in line with the eﬀects of the C5-

pyrimidine in the 3′-unsubstituted naphthyridone (compound

67 in our preceding article).⁶

Compound 38 almost matched our ATAD2 TR-FRET target

activity and possessed even greater activity when measured

against endogenous ATAD2 in the Bromosphere assay (Table

4). It also met our probe criteria of 2 logs of selectivity over

BRD4 BD1. While this was encouraging, concerns remained

over the polarity of the C3′ cyclic sulfone and its possible

impact on cell permeability. For early analogues such as 3 that

retained signiﬁcant BRD4 BD1 potency, IL-6 inhibitory activity

in LPS-stimulated PBMCs was consistent with this BET-driven

readout, showing that they were cell-permeable.⁶Less polar

analogues, such as C3′-cyclohexyl 20 or tetrahydropyran

derivatives 37 and 39, possess measurable artiﬁcial membrane

permeability (Table 4) and also have expected levels of BET-

driven IL-6 activity (Table S1, Supporting Information).

However, these have insuﬃcient ATAD2 potency and/or

BET selectivity to meet our probe criteria. For selective

ATAD2 inhibitors such as 36, 38 and 40, BET-driven cellular

IL-6 activity would be expected to be minimal, so this could no

longer be used as an indication of cell penetration. The low

logD and artiﬁcial membrane permeability of these sulfone-

containing compounds (Table 4) did suggest that their

concentration within cells could be relatively limited.

Increasing Permeability. Because of these concerns, we

attempted to increase the permeability of the sulfone-bearing

inhibitors by reducing the polar surface area and the number of

hydrogen bond donors and acceptors. In parallel, we tried to

increase logD by ﬁnding areas where small lipophilic

substituents could be tolerated. Table 5 highlights some results

of this strategy. First, to remove one heteroatom, the

naphthyridone core was changed back to the quinolinone (as

in the original hit 1). The resulting compounds included the

direct quinolinone analogue of 38 (the racemic 42 and its most

potent single enantiomer, 43). Although the removal of the

naphthyridone nitrogen did increase logD by ∼0.5, this did not

translate into measurable artiﬁcial membrane permeability.

A crystal structure of 42 was solved in ATAD2. As before, the

(R,R) enantiomer was observed to bind (Figure S3, Supporting

Information). The binding mode is very similar to that of 30

(Figure 5). The key protein contacts made by the quinolinone

of 42 and the naphthyridone of 30 are essentially identical. The

piperidines of each compound also overlay perfectly. The

increase of potency conferred by the C5-(3-pyridyl) group can

be rationalized in the same way as that previously used to

explain the increase in potency for 3 over 2.⁶The pyridine

nitrogen forms a direct hydrogen bond with the backbone NH

group of Asp1014 in the ZA loop of ATAD2 (Figure 5).

On the RVF shelf, the hydrogen bonds between Arg1077 and

the sulfone of 42 are also the same as seen in the complex with

30 (Figure 5). Unexpectedly, the side chain of Arg1007 moves

away from the sulfone in the complex with 42, suggesting that

Figure 4. (A−C) Three diﬀerent views of the X-ray structure of

ATAD2 bound to 30 (cyan, PDB code 5a82), superimposed on 2

(orange, PDB code 5a5q). In (C), a water molecule H₂O_(R1077)is

shown. This lies near Arg1077 in the complex with 2 and is displaced

by 30.

the direct C5-H analogues (Table 4: compare 20 to 17, 37 to

29, and 38 to 32). As was found for the 5-H analogues, the

most potent C5-(3-pyridyl)-substituted compounds were the

C3′-cyclic sulfones (36, 38, and 40, all with ATAD2 pIC₅₀6.9).

The 3′ tetrahydopyran derivatives were uniformly slightly lower

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a

Table 4. Combinations of Optimal C5 and C3′ Substituents

a

All are the most potent single enantiomers. Δ = diﬀerence between ATAD2 and BRD4 BD1 FRET pIC₅₀. For statistics, see Table S1, Supporting

Information

a

Table 5. Increasing Lipophilicity in the Sulfone Series

ATAD2

pIC₅₀

ATAD2 ATAD2 Bromophere BRD4 BD1

Solubility

μM

Permeability (nm/s,

pH 7.4)

Compound

X

Y

Z

R

LE

pIC₅₀

Δ

LogD

38

42

43

44

45

46

N

H

CH₃

C₂H₅

CH₃

H

6.9

6.8

7.2

7.0

7.1

6.5

0.26

0.27

0.25

0.26

0.24

7.5

7.4

7.2

7.3

7.0

4.8

5.4

4.6

5.0

4.1

2.1

1.4

1.8

2.4

2.1

2.4

179

1.6

2.0

2.1

1.7

1.8

2.3

<3

<10

CH

N

H

217

H

≥266

30

CH₃

H

N

H

323

N

H

CH₃

≥341

a

All are single (R,R) enantiomers except for 42, which is a racemic mixture of trans enantiomers. Δ = diﬀerence between ATAD2 and BRD4 BD1

FRET pIC₅₀. LE ≈ 1.37 × pIC₅₀/(number of heavy atoms). For statistics, see Table S1, Supporting Information.

their direct interaction seen in the complex with 30 is not

critical for sulfone recognition.

some positions on the C3′ substituent with no impact on

activity or selectivity. For example, we tried to mask some of

the polarity of the cyclic sulfone by methylation upon either

side (44). This change was tolerated in terms of potency, but it

did not aﬀect measured logD or permeability, and it was

somewhat detrimental to solubility, which was otherwise good

in this series. Perhaps more surprisingly, replacement of the

naphthyridone C3-methyl group, which binds in the acetyl-

lysine methyl pocket, with a bulkier ethyl substituent (45) was

also well tolerated. Unfortunately, this did not improve the

measured permeability. Finally, small piperidine N-alkyl

substituents were introduced. Piperidine N-methylation led to

a modest loss of potency at ATAD2 for compound 46 relative

The ATAD2 activity of 43, the (R,R) enantiomer of 42, is

similar to that of 38 (Table 5). This is as expected given the

lack of any visible interactions in the X-ray structures involving

the naphthyridone N7 atom or the corresponding quinolinone

C7 atom. For reasons not well understood, a small but

consistent decrease in selectivity over BET was observed for the

quinolinones relative to the naphthyridones (compare 43 to 38,

and other analogues not shown).

Finding no advantage in the quinolinone series over the

naphthyridones, we investigated the addition of small lipophilic

substituents elsewhere in the structure. These were tolerated at

H

DOI: 10.1021/acs.jmedchem.5b00773

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Journal of Medicinal Chemistry

Article

Figure 5. (A, B) Two orientations of the ATAD2 X-ray complex with 42 (green, PDB code 5a83), superimposed on the structure of 30 (cyan, PDB

code 5a82). The position of the mobile side chain of Arg1007 in the complex with 30 is shown in magenta.

to 38, probably due to the close proximity of the N-methyl

group to the side chain of Asp1071 (Figure 5). A similar

reduction was seen in the Bromosphere assay (Table 5).

Although not quite reaching our ATAD2 target potency, 46

had higher logD than 38 and lower BRD4 BD1 activity,

resulting in a slightly wider selectivity window.

Further Characterization. This work was primarily driven

by activity against the ATAD2 bromodomain, measured in the

TR-FRET assay, as well as in the Bromosphere assay as

described above. For some compounds, pK_dvalues were also

derived by monitoring direct binding to the ATAD2

bromodomain using surface plasmon resonance (SPR). SPR

pK_dvalues were in excellent agreement with the TR-FRET

pIC₅₀s (r²> 0.95, Table 6 and Figure S1c, Supporting

Table 6. ATAD2 SPR Binding Constants Compared to TR-

FRET pIC₅₀Values

ATAD2 SPR K_d

μM

ATAD2 SPR

ATAD2 TR-FRET

pIC₅₀

Compound

pK_d

13

17

22

29

32

38

43

46

2.9

5.5

5.6

7.3

5.5

6.0

7.1

7.2

6.7

5.5

5.6

6.9

5.4

6.1

6.9

7.2

6.5

2.3

0.05

3.2

1.1

0.09

0.06

0.20

Figure 6. Selectivity of 46 against other bromodomains. The color

scale ranges from pK_i7.7 (red) to <4.3 (blue, triangles denote “<”

modiﬁer).

Information). This conﬁrmed the sub-100 nM (pIC₅₀≥ 7.0)

potency of key molecules such as 38. Much of the important

SAR also tracked, for example, the decrease in potency caused

by the piperidine N-methylation of 38 to give 46.

The selectivity of 46 against the wider bromodomain family

was assessed in the BROMOscan panel (Figure 6 and Table S2,

Supporting Information).¹⁵The ATAD2 pK_iof 7.7 (K_i20 nM)

in this format was slightly more potent than in other assays.

Similar activity was found against the closest homologue of

ATAD2, ATAD2B (pK_i7.4), which was expected given the

high sequence similarity between the two bromodomains (73%

identity, with only conservative changes in the KAc site, see

Figure S2, Supporting Information). Selectivity over the BET

bromodomains was conﬁrmed, with a window of at least 2.6

logs (>400-fold), BRD4 BD1 being the most potent (pK_i5.1).

For the other bromodomains tested, the highest activity was

with the second bromodomains of TAF1 and TAF1L (pK_i7.3

and 6.9, respectively). Weaker activity remained against the

BRPF family (pK_i5.3/6.4/6.1 against BRPF1/2/3, respec-

tively) and CECR2 (pK_i6.1). The cellular consequences of

inhibition of these bromodomains is currently unknown, so we

I

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Journal of Medicinal Chemistry

Article

Figure 7. Selectivity of (A) 20, (B) 43, and (C) 46 for ATAD2 against other bromodomain containing proteins using MS-detected pull downs from

cell lysates. Speciﬁed values are apparent pK_ds. Values <4 indicate that no pK_dwas determined up to 100 μM compound concentration. No bar

indicates that protein was not identiﬁed.

Table 7. Summary of Properties of 38 and 46

38

46

ATAD2 pIC₅₀(TR-FRET)

6.9

7.5

7.1

6.5

6.6

ATAD2 pIC₅₀(Peptide FRET)

ATAD2 pIC₅₀(Bromosphere)

ATAD2 pK_d(SPR)

7.0

6.7

ATAD2 pK_i(BROMOscan)

7.7

BRD2 BD1/BD2 pIC₅₀(TR-FRET)

BRD3 BD1/BD2 pIC₅₀(TR-FRET)

BRD4 BD1/BD2 pIC₅₀(TR-FRET)

BRDT BD1/BD2 pIC₅₀(TR-FRET)

Chrom LogD (pH 7.4)

4.5/<4.5

<4.3/<4.3

4.8/5.3

4.5/<4.3

1.6

<4.3/<4.3

4.1/<3.3

<4.3/< 4.3

2.3

Artiﬁcial membrane permeability (nm/s, pH 7.4)

CLND solubility (μM)

<3

<10

179

≥341

were satisﬁed to ﬁnd this highly restricted oﬀ-target proﬁle

among the bromodomain family and to verify the excellent

selectivity over the BET bromodomains.

CONCLUSIONS

■

Here, we have presented the ﬁrst report of successful

optimization of inhibitors of the ATAD2 bromodomain to

sub-100 nMaﬃnity. Potency and BET selectivity were

improved with the help of extensive crystallography in

ATAD2 and BRD4 BD1. This led to the identiﬁcation of

compounds such as 38 (Table 7), which matched our potency

and selectivity criteria, showing >100-fold selectivity over the

BET bromodomains. This selectivity was achieved by

optimizing the 3′ substituent for complementarity with the

ATAD2 site and against the BET site, resulting in the C3′-

cyclic sulfone moiety found within 38. However, this came at

the expense of greater hydrophilicity, and the passive

permeability of the sulfone-containing members of this series

remained concerningly low for use in cellular assays. We were

partly able to mitigate this in compound 46, although the

potency was lower than we had hoped and some oﬀ-target

activity remained against other bromodomains. The conclusion

In parallel, we assessed the selectivity of 46 and other

examples against a panel of endogenous bromodomain-

containing proteins present in cell lysates using a Bromosphere

chemoproteomic assay. A focused panel of immobilized,

unselective bromodomain inhibitors was able to pull down 19

of these proteins, as detected using mass spectrometry. The

pK_dvalues of 20, 43, and 46 for those full-length proteins were

determined using competition dose−response experiments. 46

shows clearly improved selectivity over 20 (Figure 7c), as also

found for the BET bromodomains by TR-FRET. For 46, the

bromodomains detected to bind most strongly are consistent

with those found in the BROMOscan panel, although,

pleasingly, it appears that the compound shows greater

selectivity for ATAD2 and ATAD2B when measured using

endogenous proteins.

J

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Journal of Medicinal Chemistry

Article

dilution steps for, in total, 10 or 11 data points. DMSO concentration

was 1% (v/v). After 2 h incubation, the nonbound fraction was

removed by washing the beads with lysis buﬀer (50 mM Tris-HCl (pH

7.4), 0.1% (v/v) Igepal-CA630, 5% (v/v) glycerol, 150 mM NaCl, 1.5

mM MgCl₂, 25 mM NaF, 1 mM sodium vanadate, 1 mM

dithiothreitol). Proteins retained on the beads were eluted in SDS

sample buﬀer (100 mM Tris (pH 7.4), 4% (w/v) SDS, 20% (v/v)

glycerol, 0.01% (w/v) bromophenol blue, 50 mM dithiothreitol) and

spotted on nitrocellulose membranes (400 nL per spot) using an

automated pin-tool liquid transfer (Biomek FX, Beckman). After

drying, the membranes were rehydrated in 20% (v/v) ethanol and

processed for detection with a speciﬁc anti-ATAD2 antibody (Sigma;

29424 (1:300) in the presence of 0.4% Tween) followed by incubation

with an IRDye 800-labeled secondary antibody for visualization (anti-

rabbit 926-32211, LICOR (1:5000)) in the presence of 0.2% Tween.

Spot intensities were quantiﬁed using a LiCOR Odyssey scanner.

Chemoproteomic Assay with Dose-Dependent Competi-

tion. Aﬃnity proﬁling assays were performed as described

previously.^16,17Brieﬂy, sepharose beads were derivatized with

nonselective bromodomain inhibitors of various chemotypes contain-

ing a primary amine handle (e.g., 0.6 mM GSK2975292 or 2 mM

GSK2893910 or 1 mM GSK3040504, structures not shown) via

amide-bond formation on activated NHS beads. Increasing concen-

trations of tested ATAD2 inhibitor compounds were spiked into HuT-

78 mixed chromatin and nuclear extracts and incubated for 45 min at 4

°C. Derivatized sepharose beads (35 μL beads per sample) were

equilibrated in lysis buﬀer and incubated with cell extract containing

compound. Beads were washed with lysis buﬀer containing 0.2% NP-

40 and eluted with 2× SDS sample buﬀer supplemented with DTT.

Mass Spectrometry and Data Analysis for Chemoproteomic

Assay. Sample preparation, labeling with TMT isobaric mass tags,

peptide fractionation, and mass spectrometric analyses were performed

essentially as described earlier.¹⁸The pooled peptide samples were

either fractionated with a reverse phase at pH 12 as described

previously¹⁹and subsequently analyzed by LC-MS with a 1D plus

(Eksigent) LC connected to a Orbitrap XL (Thermo Scientiﬁc) or

directly analyzed using Nanoacquity UPLC system (Waters)

connected to an Orbitrap Elite mass spectrometer (Thermo

Scientiﬁc). In both cases, the peptides were separated on custom-

made 50 cm × 100 μM reversed-phase columns (Reprosil) at 40 °C.

Gradient elution was performed from 2 to 40% acetonitrile in 0.1%

formic acid over 2 h. A targeted data acquisition strategy was applied as

described²⁰for protein identiﬁcation using a list of 20 peptide charge

mass combinations per bromodomain protein. Mascot 2.3.02 (Matrix

Science) was used for protein identiﬁcation, using 10 ppm mass

tolerance for peptide precursors and 20 mDa (HCD) or 0.8 Da (CID)

mass tolerance for fragment ions. Carbamidomethylation of cysteine

residues and TMT modiﬁcation of lysine residues were set as ﬁxed

modiﬁcations, and methionine oxidation, N-terminal acetylation of

proteins, and TMT modiﬁcation of peptide N-termini were set as

variable modiﬁcations. The search database consisted of a customized

version of the IPI protein sequence database combined with a decoy

version of this database created using a script supplied by Matrix

Science. All identiﬁed proteins were quantiﬁed; FDR for quantiﬁed

proteins was ≪0.1%. For dose-dependent experiments, dose−

response curves were ﬁtted using R (http://www.r-project.org/) and

the drc package (http://www.bioassay.dk), as described previously.²¹

IC₅₀values were corrected for the inﬂuence of the immobilized ligand

on the binding equilibrium using the Cheng−Prusoﬀ relationship.²²

ATAD2 Crystallography. Apo crystals of ATAD2 981−1108,

expressed and detagged as described previously,⁶were typically grown

at 4 °C in 96-well MRC plates using well solutions under a range of

conditions spanning 0.1 M Tris-HCl, pH 7.0−8.0, 1.2−1.5 M

ammonium sulfate, and 20−25% PEG3350. The sitting drops were

made up of 3:1 ratio of protein/well solution, and the hexagonal

crystals often grew from phase-separated drops. Compounds were

soaked into apo crystals overnight either from solid or using 1−5 mM

ligand solution diluted from a 100−200 mM DMSO stock solution.

Soaked crystals were harvested in a cryo-loop, brieﬂy transferred into a

cryoprotectant buﬀer of 20% ethylene glycol + 80% well solution,

of the optimization of this series to generate a cell-permeable

and signiﬁcantly more selective ATAD2 chemical probe will be

the subject of our next publication.

EXPERIMENTAL SECTION

■

Protein Expression, SPR, BET Bromodomain Assays, PBMC

IL6 Assay, and Physicochemical Property Measurement. These

were carried out as described previously.⁶

ATAD2 Synthetic Ligand Competition TR-FRET Binding

Assay. Compounds were titrated from 10 mM in 100% DMSO,

and 100 nL was transferred to a low-volume black 384-well microtiter

plate using a Labcyte Echo 555. A Thermo Scientiﬁc Multidrop Micro

was used to dispense 5 μL of 5 nM FLAG-6His-Tev-ATAD2(981−

1121) in 50 mM Hepes, 150 mM NaCl, 5% Glycerol, 1 mM CHAPS,

and 1 mM DTT, pH 7.4, in the presence of 100 nM Alexa Fluor 488-

labeled ligand. After equilibrating for 30 min in the dark at room

temperature, the ATAD2 protein−ﬂuorescent ligand interaction was

detected using TR-FRET following a 5 μL addition of 1.5 nM

Lanthascreen Elite Tb-anti His antibody (Invitrogen, PV5863) in assay

buﬀer. Time-resolved ﬂuorescence (TRF) was then detected on a TRF

laser equipped PerkinElmer Envision multimode plate reader

(excitation = 337 nm; emission 1 = 520 nm; emission 2 = 495 nm;

dual wavelength bias dichroic = 400, 505 nm). TR-FRET ratio was

calculated using the following equation: ratio = ((acceptor

ﬂuorescence at 520 nm)/(donor ﬂuorescence at 495 nm)) × 1000.

TR FRET ratio data was normalized to a mean of 16 replicates per

microtiter plate of both 10 μM GSK3190320 (a potent naphthyridone

ATAD2 inhibitor similar to 37; see Figure S4, Supporting

Information) and 1% DMSO controls, and IC₅₀values were

determined for each of the compounds tested by ﬁtting the

ﬂuorescence ratio data to a four-parameter model, y = a + ((b −

a)/(1 + (10^x/10^c)^d), where a is the minimum, b is the Hill slope, c is

the IC₅₀, and d is the maximum.

Preparation of Cell Fractions for Chemoproteomic Proﬁling.

Nuclear extract was produced from HuT78 cells grown at 1 × 10⁶to 5

× 10⁶cells/mL in spinner ﬂasks. Cells were collected by

centrifugation, washed with PBS, and resuspended in three volumes

with hypotonic buﬀer A (10 mM Tris-Cl, pH 7.4, 1.5 mM MgCl₂, 10

mM KCl, 25 mM NaF, 1 mM Na₃VO₄, 1 mM DTT, and 1 Roche

protease inhibitor tablet per 25 mL). After 15 min, cells were

homogenized with a Dounce homogenizer. Nuclei were collected by

centrifugation (2500g), washed with hypotonic buﬀer A, and

homogenized in one volume of extraction buﬀer B (50 mM Tris-Cl,

pH 7.4, 1.5 mM MgCl₂, 20% glycerol, 420 mM NaCl, 25 mM NaF, 1

mM Na₃VO₄, 1 mM DTT, 400 units/mL DNase I, and 1 Roche

protease inhibitor tablet per 25 mL). Extraction was allowed to

proceed under agitation for 30 min at 4 °C before the extract was

clariﬁed by centrifugation at 13 000g. The extract was diluted in buﬀer

D (50 mM Tris-Cl, pH 7.4 (RT), 1.5 mM MgCl₂, 25 mM NaF, 1 mM

Na₃VO₄, 0.6% NP-40, 1 mM DTT, and Roche protease inhibitors),

and aliquots were snap-frozen in liquid nitrogen and stored at −80 °C.

Tight chromatin-associated protein-enriched fractions were prepared

by resuspending the remaining pellet in 10 volumes of high-salt

extraction buﬀer (20 mM Hepes, pH 7.4, 1.5 mM MgCl₂, 1 M KCl,

10% glycerol, 0.1% NP40, 0.5 mM DTT, and Roche protease

inhibitors) with 8−10 cycles of 10 s on/50 s oﬀ sonication with an

ultrasound homogenizer (Bandelin Sonoplus). After sonication and

incubation for 45 min at 4 °C, the homogenate was clariﬁed at 8000g.

The salt concentration was adjusted to 150 mM KCl through stepwise

dialysis before ultracentrifugation at 100 000g for 20 min. Aliquots

were snap-frozen in liquid nitrogen and stored at −80 °C.

ATAD2 Bromosphere Assay.¹⁶The ATAD2 Bromosphere

competition binding assay was performed in a 384-well format using

an antibody-based array readout. Brieﬂy, 60−120 μg HuT-78

chromatin lysate and 1.5 μL of ATAD2 capturing matrix (immobilized

GSK2998821 on NHS Sepharose, structure not shown) per well (ﬁnal

volume 75 μL) were incubated in the absence or presence of inhibitor

compound at 4 °C. Compounds were tested in a dose−response

format with a starting concentration of 100−200 μM, applying 1:3

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before being plunge-frozen in liquid nitrogen. Data from single crystals

were collected at 100 K on an in-house Rigaku FR-E+ SuperBright/

Saturn A200 detector/ACTOR robotic system or at the Diamond

Light Source (Oxford) or the Europeon Synchrotron Radiation

Facility (Genoble). Data processing was performed using DENZO,²³

MOSFLM,²⁴or XDS²⁵(within AUTOPROC [Global Phasing

Limited]) and scaled using either SCALEPACK,²³SCALA,²⁶or

AIMLESS²⁷[within the CCP4 programming suite²⁸]. Structures were

solved by Fourier synthesis using REFMAC²⁹(via CCP4) starting

from a previously determined in-house structure; model-building was

performed using COOT,³⁰and reﬁnement, using REFMAC via CCP4.

The statistics for the data collection and reﬁned coordinates are given

in Table S3, Supporting Information, and OMIT maps are provided in

Figure S3. The ﬁnal crystal structures have been deposited in the

Protein Data Bank under the accession codes shown in Table S3,

Supporting Information.

BRD4 BD1 Crystallography. Detagged BRD4-BD1(44−168),

generated as previously described,³¹was cocrystallized with at least 3:1

excess of compound at 9−10 mg/mL in sitting drops using a 96-well

MRC plate with a well solution indicated in Table S4. Crystals were

brieﬂy transferred into a cryoprotectant solution consisting of well

solution with 20% ethylene glycol added prior to ﬂash freezing in

liquid nitrogen. Data from single crystals at 100 K on an in-house

Rigaku FR-E⁺SuperBright/Saturn A200 detector/ACTOR robotic

system or at the Europeon Synchrotron Radiation Facility (Genoble)

were processed using XDS²⁵and scaled using SCALA²⁶or

AIMLESS²⁷within the CCP4 programming suite.²⁸Molecular

replacement solutions were determined with a previously collected

in-house structure. The P2₁2₁2₁cells have a single molecule in the

asymmetric unit. Manual model building was performed using

COOT³⁰and reﬁned using REFMAC²⁹via CCP4. There was clear

diﬀerence density for the ligands in the acetylated lysine binding site,

allowing the ligand to be unambiguously modeled. The statistics for

the data collection and reﬁned coordinates are given in Table S4,

Supporting Information, and electron density maps are shown in

Figure S3, Supporting Information. The ﬁnal crystal structures have

been deposited in the Protein Data Bank under the accession codes

shown in Table S3, Supporting Information.

Table 8. LCMS Gradients Employed

time (min)

ﬂow rate (mL/min)

% A

% B

0

1

99

3

1

1.5

1.9

2.0

97

100

3

0

ionization mode, alternate-scan positive and negative electrospray;

scan range, 100−1000 AMU; scan time, 0.27 s; inter scan delay, 0.10 s.

MDAP. Formate method: HPLC analysis was conducted on either a

Sunﬁre C18 column (100 mm × 19 mm, i.d. 5 μm packing diameter)

or a Sunﬁre C18 column (150 mm × 30 mm, i.d. 5 μm packing

diameter) at ambient temperature. The solvents employed were A =

0.1% v/v solution of formic acid in water and B = 0.1% v/v solution of

formic acid in acetonitrile. Run as a gradient over either 15 or 25 min

(extended run) with a ﬂow rate of 20 mL/min (100 mm × 19 mm, i.d

5 μm packing diameter) or 40 mL/min (150 mm × 30 mm, i.d. 5 μm

packing diameter). High-pH method: HPLC analysis was conducted

on either an Xbridge C18 column (100 mm × 19 mm, i.d. 5 μm

packing diameter) or a Xbridge C18 column (100 mm × 30 mm, i.d. 5

μm packing diameter) at ambient temperature. The solvents employed

were A = 10 mM ammonium bicarbonate in water, adjusted to pH 10

with ammonia solution, and B = acetonitrile. Run as a gradient over

either 15 or 25 min (extended run) with a ﬂow rate of 20 mL/min

(100 mm × 19 mm, i.d. 5 μm packing diameter) or 40 mL/min (100

mm × 30 mm, i.d. 5 μm packing diameter). For both methods, the UV

detection was a summed signal from wavelength of 210 to 350 nm.

Mass spectra were obtained on a Waters ZQ instrument; ionization

mode, alternate-scan positive and negative electrospray; scan range,

100−1000 AMU; scan time, 0.50 s; inter scan delay, 0.20 s.

(3R,4R)-tert-Butyl 4-Azido-3-hydroxypiperidine-1-carboxy-

late (5). A solution of tert-butyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-

carboxylate (4¹³) (11 g, 55 mmol) in DMF (100 mL) at room

temperature was treated with a solution of sodium azide (6 g, 92

mmol) in water (40 mL) and acetone (60 mL), and the resulting

mixture was stirred at 80 °C for 2 h and was then cooled to room

temperature and diluted with EtOAc (200 mL) and water (200 mL).

The layers were separated, and the organic phase was washed with

water (200 mL), dried over MgSO₄, and concentrated in vacuo to give

a pale yellow liquid. Puriﬁcation of this residue by ﬂash

chromatography on silica gel (340 g column, gradient: 0 to 50%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-azido-3-hydroxypi-

peridine-1-carboxylate (8.4 g, 63%) as a white solid. Later eluting

fractions were combined and concentrated in vacuo to give (3R,4R)-

tert-butyl 3-azido-4-hydroxy piperidine-1-carboxylate (1.9 g, 14%) as a

Chemistry. All solvents were purchased from Sigma-Aldrich (Hy-

Dry anhydrous solvents), and commercially available reagents were

used as received. All reactions were followed by TLC analysis (TLC

plates GF254, Merck) or LCMS (liquid chromatography mass

spectrometry) using a Waters ZQ instrument. NMR spectra were

recorded on a Bruker nanobay 400 MHz or a Bruker AVII+ 600 MHz

1

spectrometers and are referenced as follows: H NMR (400 or 600

MHz), internal standard TMS at δ = 0.00; ¹³C NMR (100.6 or 150.9

MHz), internal standard CDCl₃at δ = 77.23 or DMSO-d₆at δ = 39.70.

Column chromatography was performed on prepacked silica gel

columns (30−90 mesh, IST) using a biotage SP4. Mass spectra were

recorded on Waters ZQ (ESI-MS) and Q-Tof 2 (HRMS)

spectrometers. Mass-directed auto prep was performed on a Waters

2767 with a MicroMass ZQ mass spectrometer using a Supelco

LCABZ++ column.

1

colorless solid. H NMR (T = 120 °C, 400 MHz, DMSO-d₆) δ ppm

4.97 (br s, 1H) 3.89−4.01 (m, 1H), 3.82 (d, J = 13.3 Hz, 1H), 3.27−

3.44 (m, 2H), 2.83−2.90 (m 1H), 2.69 (dd, J = 12.8, 9.1 Hz, 1H),

1.84−2.01 (m, 1H), 1.44 (s, 9H), 1.27−1.40 (m, 1H). The racemic

mixture was separated by chiral HLPC. The HPLC analysis was carried

out on a Chiralpak AD-H (4.6 mm i.d. × 25 cm), using 20% EtOH in

heptane at a ﬂow rate of 1 mL/min. The material (4.9 g) was dissolved

in a mixture of EtOH/heptane (1:1) (500 mg in 2 mL). The

puriﬁcation was carried out on a Chiralpak AD-H (30 mm × 25 cm),

using 20% EtOH in heptane at a ﬂow rate of 30 mL/min, and 2 mL of

solution was injected at a time. The appropriate fractions were

combined and concentrated under reduced pressure to give the fastest

running enantiomer, (3R,4R)-tert-butyl 4-azido-3-hydroxypiperidine-1-

carboxylate (5) (2.3 g), and the slowest running enantiomer, (3S,4S)-

tert-butyl 4-azido-3-hydroxypiperidine-1-carboxylate (2.4 g).

Synthetic Methods and Characterization of Compounds.

Abbreviations for multiplicities observed in NMR spectra: s; singulet;

br s, broad singulet; d, doublet; t, triplet; q, quadruplet; p, pentuplet;

spt, septuplet; m, multiplet. The purity of all compounds was

determined by LCMS and ¹H NMR and was always >95%. See

Supporting Information.

LCMS. UPLC analysis was conducted on an Acquity UPLC BEH

C18 column (50 mm × 2.1 mm, i.d. 1.7 μm packing diameter) at 40

°C. Formate method: solvents employed were A = 0.1% v/v solution

of formic acid in water and B = 0.1% v/v solution of formic acid in

acetonitrile. High-pH method: the solvents employed were A = 10

mM ammonium hydrogen carbonate in water adjusted to pH 10 with

ammonia solution and B = acetonitrile. For both methods, the gradient

employed is shown in Table 8.

8-Chloro-3-methyl-1,7-naphthyridin-2(1H)-one (7a) and 2-

(Benzyloxy)-8-chloro-3-methyl-1,7-naphthyridine (7b). The

synthesis of these two compounds is described in the preceding

article.⁶

8-(((3R,4R)-3-Methoxypiperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (9). Step 1: A solution of (3R,4R)-tert-

butyl 4-azido-3-hydroxypiperidine-1-carboxylate (5) (0.54 g, 2.23

mmol) in DMF (10 mL) at 0 °C under nitrogen was treated with

NaH (60% w/w in mineral oil, 0.116 g, 2.90 mmol), and the resulting

The UV detection was a summed signal from wavelength of 210 to

350 nm. Mass spectra were obtained on a Waters ZQ instrument;

L

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mixture was stirred at this temperature for 20 min. Iodomethane

(0.153 mL, 2.45 mmol) was then added, and the solution was stirred

for 16 h at room temperature. The mixture was then diluted with water

(20 mL), and the aqueous phase was extracted with EtOAc (20 mL).

The organic phase was washed with water (20 mL), dried over

MgSO₄, and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 50% EtOAc

in cyclohexane) gave (3R,4R)-tert-butyl 4-azido-3-methoxypiperidine-

1-carboxylate (0.48 g, 84%) as a colorless oil. Step 2: (3R,4R)-tert-

butyl 4-azido-3-methoxypiperidine-1-carboxylate (0.45 g, 1.76 mmol)

was dissolved in MeOH (40 mL) and hydrogenated in the H-Cube (1

mL/min, 10% Pd/C cartridge, full H₂mode), and then MeOH was

concentrated in vacuo to give (3R,4R)-tert-butyl 4-amino-3-methox-

ypiperidine-1-carboxylate (6, R₁Me) (0.38 g, 94%) as a colorless oil.

Step 3: A suspension of 8-chloro-3-methyl-1,7-naphthyridin-2(1H)-

one (7a) (0.08 g, 0.41 mmol), (3R,4R)-tert-butyl 4-amino-3-

methoxypiperidine-1-carboxylate (6, R₁Me) (0.156 mL, 0.617

mmol), and Caddick catalyst (0.024 g, 0.041 mmol) in THF (2

mL) in a microwave vial at room temperature under nitrogen was

treated with LiHMDS (1N in THF, 1.23 mL, 1.23 mmol), and the

resulting mixture was stirred at 120 °C for 30 min under microwave

irradiation and then cooled to room temperature. The mixture was

diluted with a saturated NH₄Cl aqueous solution (10 mL), and the

aqueous phase was extracted with EtOAc (2 × 10 mL). The combined

organic phases were dried over MgSO₄and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (25 g

column, gradient: 0 to 100% EtOAc in cyclohexane) gave (3R,4R)-tert-

butyl 3-methoxy-4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-

yl)amino)piperidine-1-carboxylate (63 mg, 39%) as a beige solid.

LCMS (high-pH method): t_R0.99 min, [M + H]⁺= 389.3. Step 4: A

solution of (3S,4S)-tert-butyl 3-methoxy-4-((3-methyl-2-oxo-1,2-dihy-

dro-1,7-naphthyridin-8-yl)amino)piperidine-1-carboxylate (76 mg,

0.20 mmol) in CH₂Cl₂(3 mL) at room temperature was treated

with TFA (1 mL), and the resulting mixture was stirred at this

temperature for 1 h and then concentrated in vacuo. Puriﬁcation of the

residue by MDAP (high-pH method) gave 8-(((3S,4S)-3-methox-

ypiperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-one (48 mg,

85%) as a pale yellow solid. The racemic mixture was separated by

chiral HLPC. The HPLC analysis was carried out on a Chiralpak AD-

H (4.6 mm i.d. × 25 cm), using 40% EtOH (+0.2% isopropylamine) in

heptane at a ﬂow rate of 1 mL/min. The material (34 mg) was

dissolved in a mixture of EtOH/heptane (2:1). The puriﬁcation was

carried out on a Chiralpak AD-H (30 mm × 25 cm), using 40% EtOH

(+ 0.2% isopropylamine) in heptane at a ﬂow rate of 30 mL/min, and

3 mL of solution was injected. The appropriate fractions were

combined and concentrated under reduced pressure to give the fastest

running enatiomer, 8-(((3R,4R)-3-methoxypiperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (9) (12 mg, 0.042 mmol, 21%),

as a white solid. LCMS (high-pH method): t_R0.62 min, [M + H]⁺=

289.2. ¹H NMR (400 MHz, CD₃OD) δ ppm 7.78 (d, J = 5.4 Hz, 1H),

7.72 (d, J = 1.0 Hz, 1H), 6.79 (d, J = 5.6 Hz, 1H), 4.12−4.20 (m, 1H),

3.45 (s, 3H), 3.37 (m, 4H), 2.99−3.06 (m, 1H), 2.69−2.78 (m, 1H),

2.51−2.58 (m, 1H), 2.24 (d, J = 1.2 Hz, 3H). Three NH not seen.

8-(((3R,4R)-3-Isopropoxypiperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (10). Step 1: a solution of (3R,4R)-tert-

butyl 4-azido-3-hydroxypiperidine-1-carboxylate (5) (0.54 g, 2.2

mmol) in DMF (10 mL) at 0 °C under nitrogen was treated with

sodium hydride (60% w/w in mineral oil, 0.116 g, 2.90 mmol), and the

resulting mixture was stirred for 20 min at this temperature. 2-

Iodopropane (0.245 mL, 2.45 mmol) was then added, and the solution

was stirred at room temperature for 16 h. The mixture was then stirred

at 70 °C for 2 h before being cooled to room temperature and treated

with water (30 mL). The aqueous phase was extracted with EtOAc (30

mL). The organic phase was then washed with water (30 mL), dried

over MgSO₄, and concentrated in vacuo. Puriﬁcation of the residue by

ﬂash chromatography on silica gel (25 g column, gradient: 0 to 50%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-azido-3-isopropox-

ypiperidine-1-carboxylate (55 mg, 9%) as a colorless oil. Step 2:

(3R,4R)-tert-butyl 4-azido-3-isopropoxypiperidine-1-carboxylate (50

mg, 0.18 mmol) was dissolved in MeOH (20 mL) and hydrogenated

in the H-Cube (1 mL/min ﬂow rate, 10% Pd/C cartridge, full H₂

mode). The solution was then concentrated in vacuo to give (3R,4R)-

tert-butyl 4-amino-3-isopropoxypiperidine-1-carboxylate (6, R₁= i-Pr)

(42 mg, 92%) as a colorless oil. Step 3: a microwave vial was charged

with 8-chloro-3-methyl-1,7-naphthyridin-2(1H)-one (7a) (0.035 g,

0.18 mmol), (3R,4R)-tert-butyl 4-amino-3-isopropoxypiperidine-1-

carboxylate (6, R₁= i-Pr) (0.053 mL, 0.19 mmol), and Caddick

catalyst (10.6 mg, 0.018 mmol) and ﬁlled with THF (2 mL), and the

resulting suspension was treated at room temperature under nitrogen

with LiHMDS (1N in THF, 0.54 mL, 0.54 mmol). The resulting

mixture was stirred at 120 °C for 30 min under microwave irradiation

and was then cooled to room temperature. The mixture was then

diluted with a saturated NH₄Cl aqueous solution (10 mL), and the

aqueous phase was extracted with EtOAc (2 × 10 mL). The combined

organic phases were dried over MgSO₄and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (25 g

column, gradient: 0 to 100% EtOAc in cyclohexane) gave 8-(((3R,4R)-

3-isopropoxypiperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-

one (12 mg, 21%) (10) as a beige solid. LCMS (high-pH method): t_R

1

0.75 min, [M + H]⁺= 317.2. H NMR (400 MHz, CD₃OD) δ ppm

7.86 (d, J = 5.6 Hz, 1H), 7.75 (d, J = 1.0 Hz, 1H), 6.88 (d, J = 5.4 Hz,

1H), 4.39 (d, J = 4.4 Hz, 1H), 4.05−4.10 (m, 1H), 3.92−4.01 (m,

1H), 3.41−3.51 (m, 2H), 3.21−3.28 (m, 1H), 3.17 (dd, J = 13.1, 4.5

Hz, 1H), 2.41−2.51 (m, 1H), 2.25 (d, J = 0.7 Hz, 3H), 1.97−2.05 (m,

1H), 1.24 (d, J = 6.1 Hz, 6H). Three NH not seen.

3-Methyl-8-(((3R,4R)-3-propoxypiperidin-4-yl)amino)-1,7-

naphthyridin-2(1H)-one (11). Step 1: a solution of (3S,4S)-tert-

butyl 4-azido-3-hydroxypiperidine-1-carboxylate (5) (1.94 g, 8.00

mmol) in DMF (30 mL) at room temperature under nitrogen was

treated with sodium hydride (60% w/w in mineral oil, 0.384 g, 9.60

mmol) and then, after 10 min, with allyl bromide (1.04 mL, 12.0

mmol). The solution was stirred at room temperature for 1 h before

being quenched with MeOH (1 mL) and diluted with water. The

aqueous phase was extracted four times with Et₂O, and the combined

organics were washed with water and brine, dried over MgSO₄, and

concentrated in vacuo to give (3S,4S)-tert-butyl 3-(allyloxy)-4-

azidopiperidine-1-carboxylate (2.32 g, 103%) as a clear oil, which

was used in the next step without further puriﬁcation. Step 2: a

solution of (3S,4S)-tert-butyl 3-(allyloxy)-4-azidopiperidine-1-carbox-

ylate (2.32 g, 8.22 mmol) in THF (40 mL) at room temperature was

treated with triphenylphosphine (3.23 g, 12.3 mmol), and the resulting

mixture was stirred at this temperature for 16 h. Water (3 mL) was

then added, and the solution was reﬂuxed for 2.5 h and then cooled to

room temperature and concentrated in vacuo. The residue was

dissolved in EtOAc, dried over MgSO₄, and concentrated in vacuo to

give a white solid. This residue was loaded on to a 50 g SCX column,

washed with MeOH, and then eluted with a 2 N NH₃solution in

MeOH. The ammoniac fractions were concentrated in vacuo to give

the desired amine still contaminated with triphenylphosphine oxide.

Puriﬁcation of this residue by ﬂash chromatography on silica gel (50 g

column, gradient: 10 to 40% (20% (2 N NH₃in MeOH) in CH₂Cl₂)

in CH₂Cl₂) gave (3S,4S)-tert-butyl 3-(allyloxy)-4-aminopiperidine-1-

carboxylate (1.45 g, 69%) as a colorless oil. Step 3: (3S,4S)-tert-butyl

3-(allyloxy)-4-aminopiperidine-1-carboxylate (300 mg, 1.17 mmol)

was dissolved in MeOH (40 mL) and hydrogenated in the H-Cube on

full hydrogen mode with 10% Pd/C cartridge at 1 mL/min ﬂow rate.

The eluent was evaporated in vacuo to give a colorless oil. The product

was redissolved in MeOH (40 mL), and hydrogenation was repeated

under the same conditions to give (3S,4S)-tert-butyl 4-amino-3-

propoxypiperidine-1-carboxylate (6, R₁= Pr) (285 mg, 94%). Step 4: a

microwave vial was charged with 8-chloro-3-methyl-1,7-naphthyridin-

2(1H)-one (7a) (0.043 g, 0.22 mmol), (3R,4R)-tert-butyl 4-amino-3-

propoxypiperidine-1-carboxylate (66 mg, 0.25 mmol), and Caddick

catalyst (0.020 g, 0.033 mmol) and was then ﬁlled with THF (2 mL).

The resulting mixture was treated with LiHMDS (1 N in THF, 0.77

mL, 0.77 mmol) and was then stirred at 80 °C for 30 min under

nitrogen and microwave irradiation before being cooled to room

temperature and diluted with CH₂Cl₂(20 mL). The organic phase was

washed with a saturated NH₄Cl aqueous solution, dried over MgSO₄,

and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

M

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chromatography on silica gel (25 g column, gradient: 0 to 10% (2 N

NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-((3-methyl-2-

oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-propoxypiperidine-1-

carboxylate (73 mg, 79%) as a brown solid. This product was dissolved

in CH₂Cl₂(2 mL) and treated at room temperature with TFA (1 mL).

The mixture was stirred at this temperature for 1 h and was then

concentrated in vacuo. The residue obtained was loaded on to a 5 g

SCX-2 cartridge, which was washed with MeOH (10 mL) and eluted

with a 2 N NH₃solution in MeOH. The ammoniac fractions were

concentrated in vacuo to give 3-methyl-8-(((3R,4R)-3-propoxypiper-

idin-4-yl)amino)-1,7-naphthyridin-2(1H)-one (11) (51 mg, 73%) as a

pale yellow solid. LCMS (high-pH method): t_R0.72 min, [M + H]⁺=

water (2 × 50 mL) and then brine (50 mL), dried over MgSO₄, and

concentrated in vacuo to give cyclohexylmethyl triﬂuoromethanesul-

fonate (20 g, 93%) as a colorless oil, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.35

(d, J = 6.1 Hz, 2H), 1.76−1.89 (m, 5H), 1.69−1.76 (m, 1H), 1.17−

1.38 (m, 3H), 0.99−1.12 (m, 2H). Step 2: a solution of (3R,4R)-tert-

butyl 4-azido-3-hydroxypiperidine-1-carboxylate (5) (15 g, 62 mmol)

in DMF (50 mL) and THF (20 mL) was cooled to 0 °C and treated

with sodium hydride (60% w/w in mineral oil, 3.2 g, 80 mmol) in

small portions over 10 min. The resulting mixture was stirred at this

temperature for 30 min. A solution of cyclohexylmethyl triﬂuor-

omethanesulfonate (20 g, 81 mmol) in THF (30 mL) was then added

dropwise over 20 min, and the resulting mixture was allowed to slowly

warm to room temperature over 2 h before being diluted with water

(500 mL). The aqueous phase was extracted with Et₂O (2 × 300 mL),

and the combined organics were washed with water (2 × 400 mL),

dried over MgSO₄, and concentrated in vacuo to give a yellow oil.

Puriﬁcation of this residue by ﬂash chromatography on silica gel (330 g

column, gradient: 0 to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-

butyl 4-azido-3-(cyclohexylmethoxy) piperidine-1-carboxylate (20.5 g,

98%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 3.97−4.34

(m, 1H), 3.84−3.94 (m, 1H), 3.36−3.38 (m, 2H), 3.32−3.39 (m, 1H),

3.10−3.20 (m, 1H), 2.84−2.96 (m, 1H), 2.60−2.83 (m, 1H), 1.88−

1.97 (m, 1H), 1.75 (t, J = 13.1 Hz, 5H), 1.53−1.64 (m, 1H), 1.48 (s,

9H), 1.11−1.45 (m, 4H), 0.97 (d, J = 11.7 Hz, 2H). Step 3: a solution

of (3R,4R)-tert-butyl 4-azido-3-(cyclohexylmethoxy)piperidine-1-car-

boxylate (20.5 g, 60.6 mmol) in THF (200 mL) at room temperature

was treated with triphenylphosphine (19.1 g, 72.7 mmol), and the

resulting solution was allowed to stand at room temperature over 2

days; then, water (30 mL) was added, and the resulting mixture was

reﬂuxed for 3 h before being cooled to room temperature and

concentrated in vacuo. The residue obtained was partitioned between

EtOAc (200 mL) and brine (200 mL), and the layers were separated.

The organic phase was dried over MgSO₄and concentrated in vacuo to

give a colorless solid. This solid was triturated with Et₂O (200 mL) for

30 min and then ﬁltered oﬀ to remove triphenylphosphine oxide. The

ﬁltrate was concentrated in vacuo. Puriﬁcation of the residue obtained

by ﬂash chromatography on silica gel (340 g column, gradient: 0 to

10% (2N NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-

amino-3-(cyclohexylmethoxy)piperidine-1-carboxylate (6, R₁= cyclo-

hexylmethyl) (14.9 g, 79%) as a pale yellow oil that crystallized on

1

317.2. H NMR (400 MHz, CDCl₃) δ ppm 7.95 (d, J = 5.4 Hz, 1H),

7.65 (d, J = 1.2 Hz, 1H), 6.69 (d, J = 5.6 Hz, 1H), 6.50 (d, J = 7.3 Hz,

1H), 4.40−4.52 (m, 1H), 3.59 (dt, J = 9.2, 6.5 Hz, 1H), 3.45−3.54 (m,

2H), 3.34−3.42 (m, 1H), 3.09−3.18 (m, 1H), 2.83 (ddd, J = 12.7,

10.1, 2.9 Hz, 1H), 2.67−2.75 (m, 1H), 2.37 (d, J = 0.7 Hz, 3H), 2.21−

2.30 (m, 1H), 1.60−1.72 (m, 1H), 1.43−1.54 (m, 2H), 0.74 (t, J = 7.5

Hz, 3H). Two NH not seen.

8-(((3R,4R)-3-Isobutoxypiperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (12). Step 1: a solution of (3R,4R)-tert-

butyl 4-azido-3-hydroxypiperidine-1-carboxylate (5) (0.70 g, 2.89

mmol) in DMF (10 mL) at 0 °C was treated with NaH (60% w/w

in mineral oil, 0.139 g, 3.47 mmol), and the resulting mixture was

stirred for 20 min at 0 °C before 1-bromo-2-methylpropane (0.396 g,

2.89 mmol) was added. The mixture was stirred at this temperature for

2 h and was then diluted with water (40 mL). The aqueous phase was

extracted with EtOAc (40 mL), and the organic layer was washed with

water (40 mL), dried over MgSO₄, and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (25 g

column, gradient: 0 to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-

butyl 4-azido-3-isobutoxypiperidine-1-carboxylate (90 mg, 10%). Step

2: a suspension of 8-chloro-3-methyl-1,7-naphthyridin-2(1H)-one (7a)

(0.050 g, 0.26 mmol), (3R,4R)-tert-butyl 4-amino-3-isobutoxypiper-

idine-1-carboxylate (0.078 g, 0.29 mmol), and Caddick catalyst (0.017

g, 0.029 mmol) in THF (2 mL) at room temperature was treated with

LiHMDS (1N in THF, 0.86 mL, 0.86 mmol), and the resulting

mixture was stirred at 120 °C under microwave irradiation for 1 h,

cooled to 0 °C, and quenched with a saturated NH₄Cl aqueous

solution (10 mL). The aqueous phase was extracted with CH₂Cl₂(10

mL). The organic layer was dried over MgSO₄and concentrated in

vacuo. Puriﬁcation of the residue by ﬂash chromatography on silica gel

(10 g column, gradient: 0 to 100% EtOAc in cyclohexane) gave

(3R,4R)-tert-butyl 3-isobutoxy-4-((3-methyl-2-oxo-1,2-dihydro-1,7-

naphthyridin-8-yl)amino)piperidine-1-carboxylate (51 mg, 41%) as a

pale yellow solid. Step 3: a solution of (3R,4R)-tert-butyl 3-isobutoxy-

4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-

piperidine-1-carboxylate (50 mg, 0.12 mmol) in CH₂Cl₂(3 mL) at

room temperature was treated with TFA (1 mL), and the resulting

mixture was stirred at this temperature for 1 h and concentrated in

vacuo. The residue was loaded on to a 5 g SCX-2 cartridge, washed

with MeOH (20 mL), and eluted with a 2 N NH₃solution in MeOH.

The ammoniac fractions were concentrated in vacuo to give 8-

(((3R,4R)-3-isobutoxypiperidin-4-yl)amino)-3-methyl-1,7-naphthyri-

din-2(1H)-one (12) (31 mg, 81%) as a pale yellow solid. LCMS (high-

1

standing overnight. H NMR (400 MHz, CDCl₃) δ ppm 4.13−4.49

(m, 1H), 3.93−4.09 (m, 1H), 3.42−3.50 (m, 1H), 3.25 (dd, J = 9.0,

6.6 Hz, 1H), 2.80−2.90 (m, 1H), 2.73 (br s, 2H), 2.31−2.53 (m, 1H),

1.64−1.87 (m, 6H), 1.53−1.63 (m, 1H), 1.43−1.50 (m, 9H), 1.10−

1.38 (m, 6H), 0.87−1.02 (m, 2H). Step 4: a ﬂask was charged with 2-

(benzyloxy)-8-chloro-3-methyl-1,7-naphthyridine (7b) (530 mg, 1.86

mmol), sodium tert-butoxide (537 mg, 5.58 mmol), Brettphos

palladacycle (74.3 mg, 0.093 mmol), Brettphos (50.0 mg, 0.093

mmol), and (3S,4S)-tert-butyl 4-amino-3-(cyclohexylmethoxy)-

piperidine-1-carboxylate (756 mg, 2.42 mmol) and then ﬁlled with

THF (10 mL), and the resulting mixture stirred was for 1 h at room

temperature. The color of the reaction changed from deep green to

orange during this time. The mixture was then diluted with EtOAc (20

mL), and the organic phase was washed with water (20 mL) and then

dried over MgSO₄, and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (50 g column, gradient: 0

to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-(cyclohexylme-

1

pH method): t_R0.80 min, [M + H]⁺= 331.3. H NMR (400 MHz,

DMSO-d₆) δ ppm 7.72 (d, J = 5.4 Hz, 1H), 7.67 (d, J = 1.2 Hz, 1H),

6.68 (d, J = 5.4 Hz, 1H), 6.62 (d, J = 7.3 Hz, 1H), 4.13 (m, 1H),

3.35(br s, 1H), 3.30 (dd, J = 8.9, 6.5 Hz, 2H), 3.13−3.23 (m, 3H),

2.81−2.90 (m, 1H), 2.44−2.50 (m, 1H), 2.25−2.35 (m, 1H), 2.11 (d, J

= 0.7 Hz, 3H), 1.97 (dd, J = 13.0, 3.7 Hz, 1H), 1.62 (dt, J = 13.2, 6.6

Hz, 1H), 1.22−1.35 (m, 1H), 0.69 (dd, J = 8.6, 6.8 Hz, 6H).

8-(((3R,4R)-3-(Cyclohexylmethoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (13). Step 1: a solution of

cyclohexylmethanol (10 g, 88 mmol) in CH₂Cl₂(150 mL) at 0 °C

under nitrogen was treated with pyridine (7.8 mL, 96 mmol), and the

resulting mixture was stirred for 5 min before being treated with the

dropwise addition of Tf₂O (16.3 mL, 96 mmol) over 10 min. The

resulting mixture was stirred for 1 h at this temperature and was then

warmed to room temperature. The organic phase was washed with

thoxy) piperidine-1-carboxylate (1.02 g, 98%) as a pale yellow solid.

1

LCMS (high-pH method): t_R1.73 min, [M + H]⁺561. H NMR

=

(400 MHz, CDCl₃) δ ppm 7.91 (d, J = 5.6 Hz, 1H), 7.67 (d, J = 1.2

Hz, 1H), 7.50 (d, J = 7.1 Hz, 2H), 7.39−7.44 (m, 2H), 7.36 (d, J = 7.3

Hz, 1H), 6.74 (d, J = 5.6 Hz, 1H), 6.37 (d, J = 7.3 Hz, 1H), 5.47−5.56

(m, 2H), 4.18−4.32 (m, 1H), 3.85 (s, 1H), 3.40−3.49 (m, 1H), 3.29−

3.37 (m, 1H), 3.22 (dd, J = 8.9, 6.5 Hz, 2H), 2.42−2.51 (m, 1H), 2.40

(d, J = 0.7 Hz, 3H), 1.47−1.66 (m, 18H), 1.05 (d, J = 8.3 Hz, 3H),

0.68−0.86 (m, 2H). Step 5: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-(cyclohexylme-

thoxy) piperidine-1-carboxylate (1.02 g, 1.82 mmol) in MeOH (100

N

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mL) was hydrogenated using a H-Cube apparatus, on full hydrogen

mode, running at 1 mL/min with a 10% Pd/C cartridge. The eluent

was evaporated in vacuo to give (3R,4R)-tert-butyl 3-(cyclohexylme-

thoxy)-4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-

piperidine-1-carboxylate (0.85 g, 99%) as a pale yellow solid which was

used in the next step without further puriﬁcation. LCMS (high-pH

method): t_R1.47 min, [M + H]⁺= 471. ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 11.06−11.22 (m, 1H), 7.76 (d, J = 5.3 Hz, 1H), 7.65 (d, J =

1.0 Hz, 1H), 6.73 (d, J = 5.3 Hz, 1H), 6.46 (d, J = 7.0 Hz, 1H), 4.29−

4.38 (m, 1H), 3.76−3.84 (m, 1H), 3.60−3.69 (m, 1H), 3.39 (dd, J =

9.3, 6.5 Hz, 1H), 3.14−3.36 (m, 4H), 2.15 (d, J = 1.0 Hz, 3H), 1.97−

2.08 (m, 1H), 1.59 (d, J = 7.5 Hz, 5H), 1.39−1.53 (m, 11H), 1.02−

1.22 (m, 3H), 0.89 (br s, 2H). Step 6: a solution of (3R,4R)-tert-butyl

3-(cyclohexylmethoxy)-4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyri-

din-8-yl)amino)piperidine-1-carboxylate (50 mg, 0.11 mmol) in

CH₂Cl₂(3 mL) at room temperature was treated with TFA (1 mL),

and the resulting mixture was stirred at this temperature for 1 h and

then concentrated in vacuo. The residue was dissolved in MeOH (5

mL) and loaded onto a 5g SCX-2 cartridge. The cartridge was washed

with MeOH (20 mL) and was then eluted with a 2 N NH₃solution in

MeOH (10 mL). The ammoniac fractions were concentrated in vacuo

to give 8-(((3R,4R)-3-(cyclohexylmethoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (13) (33 mg, 84%) as a yellow

The organic layer was dried over Na₂SO₄and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (10 g

column, gradient: 0 to 100% EtOAc in cyclohexane) gave (3R,4R)-tert-

butyl 3-(cyclopentylmethoxy)-4-((3-methyl-2-oxo-1,2-dihydro-1,7-

naphthyridin-8-yl)amino)piperidine-1-carboxylate (145 mg, 95%) as

a yellow glass. A solution of (3R,4R)-tert-butyl 3-(cyclopentylme-

thoxy)-4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-

piperidine-1-carboxylate (145 mg, 0.32 mmol) in CH₂Cl₂(3 mL) at

room temperature was treated with TFA (1 mL), and the resulting

mixture was stirred at room temperature for 1 h and then concentrated

in vacuo. The residue was loaded on to a 5 g SCX-2 cartridge, washed

with MeOH, and eluted with a 2 N NH₃solution in MeOH. The

ammoniac fractions were combined and concentrated in vacuo to give

8-(((3R,4R)-3-(cyclopentylmethoxy)piperidin-4-yl)amino)-3-methyl-

1,7-naphthyridin-2(1H)-one (14) (110 mg, 92%) as a yellow solid.

1

LCMS (high-pH method): t_R0.89 min, [M + H]⁺= 357. H NMR

(400 MHz, CDCl₃) δ ppm 7.96 (d, J = 5.4 Hz, 1H), 7.66 (s, 1H), 6.69

(d, J = 5.4 Hz, 1H), 6.48 (d, J = 7.1 Hz, 1H), 4.51−4.40 (m, 1H),

3.54−3.43 (m, 2H), 3.42−3.32 (m, 2H), 3.17−3.07 (m, 1H), 2.87−

2.77 (m, 1H), 2.74−2.65 (m, 1H), 2.38 (s, 3H), 2.30−2.21 (m, 1H),

2.11−1.97 (m, 1H), 1.73−1.61 (m, 1H), 1.59−1.43 (m, 2H), 1.43−

1.30 (m, 4H), 1.13−1.01 (m, 2H). Two NH not seen.

8-(((3R,4R)-3-(Cycloheptylmethoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (15). Step 1: a solution of

cycloheptane carboxylic acid (3 g, 21 mmol) in THF (50 mL) was

cooled to 0 °C using an ice bath and was then treated under nitrogen

with LAH (1 N in THF, 36 mL, 36 mmol) dropwise over 10 min. The

resulting mixture was stirred at this temperature for 1 h and was then

stirred at room temperature for 16 h. The reaction mixture was

quenched by the dropwise addition of water (1.4 mL) over 10 min,

followed by a 15% w/w NaOH aqueous solution (1.4 mL). After

stirring for 20 min, the mixture was ﬁltered, and the ﬁltrate was

concentrated in vacuo to give cycloheptylmethanol (2.55 g, 94%) as a

colorless oil. Step 2: cycloheptylmethyl triﬂuoromethanesulfonate was

prepared from cycloheptylmethanol using the same procedure as that

for the synthesis of cyclopentylmethyl triﬂuoromethanesulfonate from

cyclopentylmethanol (preparation of 14, step 1) (5.2 g, 95%, colorless

oil). Step 3: (3R,4R)-tert-butyl 4-azido-3-(cycloheptylmethoxy)

piperidine-1-carboxylate was prepared from cycloheptylmethyl tri-

ﬂuoromethanesulfonate and (3R,4R)-tert-butyl 4-azido-3-hydroxypi-

peridine-1-carboxylate (5) using the same procedure as that for the

synthesis of (3R,4R)-tert-butyl 4-azido-3-(cyclopentylmethoxy)-

piperidine-1-carboxylate from cyclopentylmethyl triﬂuoromethanesul-

fonate (preparation of 14, step 2) (3.4 g, 78%, colorless oil). Step 4:

(3R,4R)-tert-butyl 4-amino-3-(cycloheptylmethoxy)piperidine-1-car-

boxylate was prepared from (3R,4R)-tert-butyl 4-azido-3-

(cycloheptylmethoxy)piperidine-1-carboxylate using the same proce-

dure as that for the synthesis of (3R,4R)-tert-butyl 4-amino-3-

(cyclopentylmethoxy)piperidine-1-carboxylate from (3R,4R)-tert-

butyl 4-azido-3-(cyclopentylmethoxy)piperidine-1-carboxylate (prep-

aration of 14, step 3). Puriﬁcation of the crude amine by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 10% (2 N

NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-amino-3-

(cycloheptylmethoxy) piperidine-1-carboxylate (1.35 g, 97%) as a

colorless oil. Step 5: 8-(((3R,4R)-3-(cycloheptylmethoxy)piperidin-4-

yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-one was prepared from

(3R,4R)-tert-butyl 4-amino-3-(cycloheptylmethoxy) piperidine-1-car-

boxylate and 8-chloro-3-methyl-1,7-naphthyridin-2(1H)-one (7a)

using the same procedure as that for the synthesis of 8-(((3R,4R)-3-

(cyclopentylmethoxy)piperidin-4-yl)amino)-3-methyl-1,7-naphthyri-

din-2(1H)-one (preparation of 14, step 4). Puriﬁcation of the crude

amine by MDAP (high-pH method) gave 8-(((3R,4R)-3-(cycloheptyl

methoxy)piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-one

(18 mg, 13%) as a yellow oil. LCMS (high-pH method): t_R1.01 min,

[M + H]⁺= 385. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.72 (d, J =

5.4 Hz, 1H), 7.67 (s, 1H), 6.67 (d, J = 5.4 Hz, 1H), 6.59 (d, J = 7.6 Hz,

1H), 4.19−4.08 (m, 1H), 3.22−3.12 (m, 3H), 2.90−2.81 (m, 1H),

2.49−2.43 (m, 1H), 2.34−2.24 (m, 1H), 2.11 (s, 3H), 1.99−1.90 (m,

1H), 1.57−1.11 (m, 13H), 1.01−0.87 (m, 2H). Two NH not seen.

1

solid. LCMS (high-pH method): t_R0.95 min, [M + H]⁺= 371.3. H

NMR (400 MHz, CDCl₃) δ ppm 13.2 (br s, 1H), 7.96 (d, J = 5.4 Hz,

1H), 7.65 (d, J = 1.0 Hz, 1H), 6.69 (d, J = 5.6 Hz, 1H), 6.45 (d, J = 6.4

Hz, 1H), 4.46 (d, J = 5.1 Hz, 1H), 3.34−3.49 (m, 3H), 3.29 (dd, J =

9.2, 6.5 Hz, 1H), 3.09−3.17 (m, 1H), 2.78−2.88 (m, 1H), 2.70 (dd, J

= 12.1, 8.4 Hz, 1H), 2.38 (d, J = 0.7 Hz, 3H), 2.20−2.28 (m, 1H),

1.62−1.77 (m, 2H), 1.39−1.61 (m, 5H), 0.88−1.13 (m, 3H), 0.65−

0.83 (m, 2H). One NH not seen.

8-(((3R,4R)-3-(Cyclopentylmethoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (14). Step 1: a solution of

cyclopentylmethanol (1.55 g, 15.5 mmol) in CH₂Cl₂(30 mL) was

cooled in an ice bath and was then treated with pyridine (1.38 mL,

17.0 mmol) followed by triﬂuoromethanesulfonic anhydride (2.88 mL,

17.0 mmol). The resulting mixture was stirred at this temperature for 2

h and was then washed with brine, dried over Na₂SO₄, and

concentrated in vacuo to give cyclopentylmethyl triﬂuoromethanesul-

fonate (3.02 g, 84%) as a colorless oil, which was used in the next step

without further puriﬁcation. Step 2: a solution of (3R,4R)-tert-butyl 4-

azido-3-hydroxypiperidine-1-carboxylate (5) (1.8 g, 7.4 mmol) in

DMF (5 mL) was cooled in an ice bath and was then treated with

NaH (60% w/w in mineral oil, 0.232 g, 9.66 mmol), and the resulting

mixture was stirred at this temperature for 20 min before being treated

with cyclopentylmethyl triﬂuoromethanesulfonate (2 g, 8.61 mmol).

After stirring at 0 °C for 2 h, the reaction mixture was allowed to warm

to room temperature. The reaction mixture was then diluted with

water (15 mL), and the aqueous phase was extracted with EtOAc (20

mL). The organic layer was washed with water (20 mL), dried over

Na₂SO₄, and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 100%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-azido-3-

(cyclopentylmethoxy)piperidine-1-carboxylate (1.01 g, 42%) as a

colorless oil. Step 3: a solution of (3R,4R)-tert-butyl 4-azido-3-

(cyclopentylmethoxy)piperidine-1-carboxylate (1.01 g, 3.11 mmol) in

MeOH (80 mL) was hydrogenated in the H-Cube on full hydrogen

mode over a 10% Pd/C cartridge at 1 mL/min ﬂow rate. The eluent

was concentrated in vacuo to give (3R,4R)-tert-butyl 4-amino-3-

(cyclopentylmethoxy) piperidine-1-carboxylate (6, R₁= cyclopentyl-

methyl) (0.92 g, 99% yield) as a colorless oil. Step 4: a microwave vial

was charged with 8-chloro-3-methyl-1,7-naphthyridin-2(1H)-one (7a)

(65 mg, 0.33 mmol), (3R,4R)-tert-butyl4-amino-3-(cyclopentylme-

thoxy) piperidine-1-carboxylate (130 mg, 0.43 mmol), and Caddick

catalyst (15 mg, 0.33 mmol) and ﬂushed with nitrogen for 10 min.

THF (2 mL) followed by LiHMDS (1N in THF, 1.34 mL, 1.34

mmol) was added, and the reaction mixture was stirred at 80 °C for 30

min under microwave irradiation and then cooled to room

temperature. The reaction mixture was diluted with CH₂Cl₂(10

mL) and washed with a saturated NH₄Cl aqueous solution (10 mL).

O

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8-(((3S,4S)-3-(Cyclohexylmethoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (16) and 8-(((3R,4R)-3-

(Cyclohexylmethoxy)piperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (17). Step 1: the racemic mixture of the

Boc-protected compound 13 (8, R₁= methylcyclohexyl, R₂= H) was

separated by chiral HLPC. The HPLC analysis was carried out on a

Chiralpak IC (4.6 mm i.d. × 25 cm), using 20% EtOH in heptane at a

ﬂow rate of 1 mL/min. The material (100 mg) was dissolved in a

mixture of EtOH/heptane (1:1) (4 mL). The puriﬁcation was carried

out on a Chiralpak IC (2 cm × 25 cm), using 20% EtOH in heptane at

a ﬂow rate of 15 mL/min, and 1 mL of solution was injected at a time.

The appropriate fractions were combined and concentrated under

reduced pressure to give the fastest running enantiomer, (3S,4S)-tert-

butyl 3-(cyclohexylmethoxy)-4-((3-methyl-2-oxo-1,2-dihydro-1,7-

naphthyridin-8-yl)amino)piperidine-1-carboxylate (28 mg, 0.059

mmol), and the slowest running enantiomer, (3R,4R)-tert-butyl 3-

(cyclohexylmethoxy)-4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyri-

din-8-yl)amino)piperidine-1-carboxylate (20 mg, 0.042 mmol). Step 2:

(3S,4S)-tert-butyl 3-(cyclohexylmethoxy)-4-((3-methyl-2-oxo-1,2-dihy-

dro-1,7-naphthyridin-8-yl)amino)piperidine-1-carboxylate (28 mg,

0.059 mmol) and (3R,4R)-tert-butyl 3-(cyclohexylmethoxy)-4-((3-

methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)piperidine-1-

carboxylate (20 mg, 0.042 mmol) were each dissolved in CH₂Cl₂(3

mL) at room temperature and then were treated with TFA (1 mL).

The mixtures were stirred at this temperature for 30 min and were

then concentrated in vacuo. The residue were dissolved in MeOH (3

mL), loaded onto a 5 g SCX-2 cartridge, washed with MeOH (10 mL),

and ﬁnally eluted with a 2 N NH₃solution in MeOH (10 mL). The

ammoniac fractions were in each case concentrated in vacuo to give 8-

(((3S,4S)-3-(cyclohexylmethoxy)piperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (16) (21 mg, 95%) as a pale yellow solid and

8-(((3R,4R)-3-(cyclohexylmethoxy)piperidin-4-yl)amino)-3-methyl-

1,7-naphthyridin-2(1H)-one (17) (15 mg, 68) as pale yellow solid.

8-(((3R,4R)-3-(Cyclohexylmethoxy)piperidin-4-yl)amino)-3-

methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one

(20). Step 1: a solution of (3R,4R)-tert-butyl 3-(cyclohexylmethoxy)-4-

((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-

piperidine-1-carboxylate (208 mg, 0.442 mmol) in CHCl₃(20 mL) at

−10 °C was treated with NBS (79 mg, 0.44 mmol), and the resulting

solution was stirred at this temperature for 1 h, treated with a saturated

sodium metabisulfate aqueous solution, and stirred for another 10 min.

The layers were separated, and the organic phase was dried using a

phase separator and concentrated in vacuo to give (3R,4R)-tert-butyl 4-

((5-bromo-3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-

3-(cyclohexylmethoxy)piperidine-1-carboxylate (232 mg, 96%) as a

pale yellow solid, which was used in the next step without further

puriﬁcation. LCMS (high-pH method): t_R1.56 min, [M + H]⁺= 549

(method formic): t_R1.11 min, [M + H]⁺= 562. ¹H NMR (400 MHz,

CDCl₃) δ ppm 13.2 (br s, 1H), 8.52 (d, J = 1.5 Hz, 1H), 8.47 (d, J =

1.7 Hz, 1H), 7.91 (s, 1H), 7.71 (d, J = 1.0 Hz, 1H), 7.53 (s, 1H),

6.66−6.77 (m, 1H), 4.50−4.65 (m, 1H), 4.22−4.46 (m, 1H), 3.98−

4.08 (m, 1H), 3.41−3.54 (m, 2H), 3.23−3.34 (m, 1H), 2.75−3.19 (m,

2H), 2.47 (s, 2H), 2.28 (d, J = 0.7 Hz, 2H), 2.20−2.26 (m, 1H), 1.99−

2.05 (m, 3H), 1.77 (br s, 3H), 1.61−1.73 (m, 3H), 1.39−1.60 (m,

14H). Step 3: a solution of (3R,4R)-tert-butyl 3-(cyclohexylmethoxy)-

4-((3-methyl-5-(5-methylpyridin-3-yl)-2-oxo-1,2-dihydro-1,7-naph-

thyridin-8-yl)amino)piperidine-1-carboxylate (65 mg, 0.12 mmol) in

CH₂Cl₂(3 mL) at room temperature was treated with TFA (1 mL),

and the resulting mixture was stirred for 1 h at this temperature and

then concentrated in vacuo. The residue was dissolved in MeOH (10

mL) and loaded onto a 5 g SCX-2 cartridge, which was washed with

MeOH (10 mL) and then eluted with a 2 N NH₃solution in MeOH.

The ammoniac fractions were combined and concentrated in vacuo.

Puriﬁcation of this residue by MDAP (high-pH method) gave 8-

(((3R,4R)-3-(cyclohexylmethoxy)piperidin-4-yl)amino)-3-methyl-5-

(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one (20) (14 mg,

26%) as a yellow solid. LCMS (high-pH method): t_R0.98 min, [M

1

+ H]⁺= 462. H NMR (400 MHz, CDCl₃) δ ppm 13.5 (br s, 1H),

8.52 (d, J = 1.5 Hz, 1H), 8.48 (d, J = 1.7 Hz, 1H), 7.91 (s, 1H), 7.71

(d, J = 1.0 Hz, 1H), 7.53 (s, 1H), 6.65−6.74 (m, 1H), 4.46−4.56 (m,

1H), 3.36−3.50 (m, 3H), 3.31 (dd, J = 9.3, 6.6 Hz, 1H), 3.14 (br s,

1H), 2.85 (br s, 1H), 2.71 (dd, J = 12.2, 8.6 Hz, 1H), 2.47 (s, 3H),

2.34 (d, J = 0.7 Hz, 3H), 2.23−2.31 (m, 1H), 1.71 (d, J = 9.8 Hz, 1H),

1.43−1.64 (m, 6H), 0.94−1.19 (m, 3H), 0.68−0.85 (m, 2H). One NH

not seen.

8-(((3S,4S)-3-(Cyclohexylmethoxy)piperidin-4-yl)amino)-3-

methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one

(21). This compound was obtained in an analogous manner as that for

compound 20, starting from (3S,4S)-tert-butyl 3-(cyclohexylmethoxy)-

4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-

piperidine-1-carboxylate.

5-(5-Aminopyridin-3-yl)-8-(((3R,4R)-3-(cyclohexylmethoxy)-

piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-one

(22). A 20 mL microwave vial was charged with (5-aminopyridin-2-

yl)boronic acid (40.2 mg, 0.291 mmol), (3R,4R)-tert-butyl 4-((5-

bromo-3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-

(cyclohexylmethoxy)piperidine-1-carboxylate (18, R₁= H, X = CH₂)

(80 mg, 0.15 mmol), K₂CO₃(80 mg, 0.582 mmol), Pd(OAc)₂(3.3

mg, 0.015 mmol), and di((3S,5S,7S)-adamantan-1-yl)(butyl)-

phosphine (cataCXium A) (5.22 mg, 0.015 mmol) and was then

ﬁlled with 1,4-dioxane (4 mL) and water (2 mL). The resulting

mixture was degassed for 20 min with nitrogen and was then stirred at

110 °C for 30 min under microwave irradiation before being cooled to

room temperature and concentrated in vacuo. The residue was

partitioned between CH₂Cl₂(20 mL) and water (20 mL), and the

layers were separated. The organic phase was dried using an

hydrophobic frit and concentrated in vacuo. Puriﬁcation of the residue

by ﬂash chromatography on silica gel (25 g column, gradient: 0 to 10%

(2 N NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-((5-(5-

aminopyridin-3-yl)-3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-

yl)amino)-3-(cyclohexylmethoxy)piperidine-1-carboxylate (57 mg,

70%) as a yellow solid. This compound was dissolved in CH₂Cl₂(3

mL), and the resulting solution was treated with TFA (1 mL). The

mixture was stirred at room temperature for 2 h and then concentrated

in vacuo. The residue was dissolved in MeOH (5 mL) and loaded onto

a 5 g SCX-2 cartridge. This cartridge was washed with MeOH (10 mL)

and then eluted with a 2 N NH₃solution in MeOH. The ammoniac

fractions were concentrated in vacuo to give 5-(5-aminopyridin-3-yl)-8-

(((3R,4R)-3-(cyclohexylmethoxy)piperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (21) (33 mg, 71%) as a yellow solid. LCMS

(method TFA): t_R0.57 min, [M + H]⁺= 463.4. ¹H NMR (400 MHz,

CD₃OD) δ ppm 8.02 (d, J = 2.7 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H),

7.68−7.74 (m, 2H), 7.12 (br s, 1H), 4.27−4.37 (m, 1H), 3.43−3.50

(m, 1H), 3.35−3.42 (m, 2H), 3.22−3.29 (m, 1H), 3.03−3.12 (m, 1H),

2.71−2.81 (m, 1H), 2.50−2.63 (m, 1H), 2.21 (d, J = 1.0 Hz, 4H), 1.54

(br s, 6H), 1.34−1.42 (m, 1H), 0.95−1.19 (m, 4H), 0.68−0.85 (m,

2H). Four NH not seen.

1

(1 Br). H NMR (400 MHz, CDCl₃) δ ppm 13.2 (br s, 1H), 8.08 (s,

1H), 8.03 (d, J = 1.0 Hz, 1H), 6.50−6.63 (m, 1H), 4.38−4.53 (m,

1H), 3.97−4.09 (m, 1H), 3.40−3.50 (m, 2H), 3.29 (d, J = 6.6 Hz,

2H), 3.02−3.18 (m, 2H), 2.36 (s, 3H), 2.15−2.26 (m, 2H), 1.48−1.64

(m, 15H), 1.07 (d, J = 12.6 Hz, 3H), 0.76 (br s, 2H). Step 2: a

microwave vial was charged with (3R,4R)-tert-butyl 4-((5-bromo-3-

methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-

(cyclohexylmethoxy)piperidine-1-carboxylate (100 mg, 0.182 mmol),

Pd(OAc)₂(4.09 mg, 0.018 mmol), cataCXium A (6.53 mg, 0.018

mmol), 5-methylpyridine-5-boronic acid (49.8 mg, 0.364 mmol), and

K₂CO₃(75 mg, 0.55 mmol) and then ﬁlled with 1,4-dioxane (3 mL)

and water (0.5 mL). The resulting mixture was stirred at 100 °C for 30

min under nitrogen and microwave irradiation and was then cooled to

room temperature. The mixture was diluted with CH₂Cl₂(10 mL),

and the organic phase was washed with water, dried over MgSO₄, and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (10 g column, gradient: 0 to 10% (2

N NH₃in MeOH) in CH₂Cl₂) gave a residue that was further puriﬁed

by ﬂash chromatography on silica gel (10 g column, gradient: 0 to 10%

(2 N NH₃in MeOH)) to give (3R,4R)-tert-butyl 3-(cyclo-

hexylmethoxy)-4-((3-methyl-5-(5-methylpyridin-3-yl)-2-oxo-1,2-dihy-

dro-1,7-naphthyridin-8-yl)amino)piperidine-1-carboxylate (19, R₁

=

H, X = CH₂, Y = C, R₂= Me) (65 mg, 64%) as a yellow gum. LCMS

P

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8-(((3R,4R)-3-((1-Acetylpiperidin-4-yl)methoxy)piperidin-4-

yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-one (25). Step 1: a

solution of piperidin-4-ylmethanol (5.00 g, 43.4 mmol) and triethyl-

amine (9.00 mL, 64.6 mmol) in CH₂Cl₂(100 mL) was cooled in an

ice bath and was then treated with acetic anhydride (4.10 mL, 43.4

mmol), and the resulting mixture was allowed to warm to room

temperature and stirred under nitrogen for 16 h. The solution was

then washed with brine (100 mL), dried over Na₂SO₄, and

concentrated in vacuo to give 1-(4-(hydroxymethyl)piperidin-1-

yl)ethanone (4.56 g, 67%) as a pale yellow gum. Step 2: a solution

of 1-(4-(hydroxymethyl)piperidin-1-yl)ethanone (2.00 g, 12.7 mmol)

and pyridine (1.24 mL, 15.3 mmol) in CH₂Cl₂(40 mL) under

nitrogen was cooled in an ice bath and then treated with

triﬂuoromethanesulfonic anhydride (2.36 mL, 14.0 mmol) dropwise

over 10 min. The resulting mixture was stirred at this temperature for

30 min and then washed with water (50 mL). The organic layer was

then dried over Na₂SO₄and concentrated in vacuo to give (1-

acetylpiperidin-4-yl)methyl triﬂuoromethanesulfonate (1.79 g, 49%) as

a brown gum, which was used in the next step without further

puriﬁcation. Step 3: a solution of (3R,4R)-tert-butyl 4-azido-3-

hydroxypiperidine-1-carboxylate (5) (1.5 g, 6.2 mmol) in DMF (5

mL) was cooled in an ice bath and then treated with NaH (60% w/w

in mineral oil, 0.19 g, 8.05 mmol), and the resulting mixture stirred at

this temperature for 20 min before (1-acetylpiperidin-4-yl)methyl

triﬂuoromethanesulfonate (1.79 g, 6.19 mmol) was added as a solution

in DMF (5 mL). The solution was stirred at 0 °C for 2 h and was then

allowed to warm to room temperature and diluted with water (15

mL). The aqueous phase was extracted with EtOAc (20 mL). The

organic layer was washed with water (20 mL), dried over Na₂SO₄, and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (10 g column, gradient: 0 to 100%

EtOAc in cyclohexane followed by 5 to 10% (2 N NH₃in MeOH) in

CH₂Cl₂) gave (3R,4R)-tert-butyl 3-((1-acetylpiperidin-4-yl)methoxy)-

4-azidopiperidine-1-carboxylate (0.43 g, 18% yield) as a yellow gum.

This azide was dissolved in MeOH (20 mL) and hydrogenated in the

H-Cube on full hydrogen mode, using a 10% Pd/C cartridge, at 1 mL/

min ﬂow rate; then, the eluent was concentrated in vacuo to give

(3R,4R)-tert-butyl3-((1-acetylpiperidin-4-yl)methoxy)-4-aminopiperi-

dine-1-carboxylate (0.34 g, 15%). Step 4: (3R,4R)-tert-butyl 3-((1-

acetylpiperidin-4-yl)methoxy)-4-aminopiperidine-1-carboxylate (225

mg, 0.632 mmol), sodium tert-butoxide (152 mg, 1.580 mmol), and

BrettPhos precatalyst (21 mg, 0.026 mmol) were added to a solution

of 2-(benzyloxy)-8-chloro-3-methyl-1,7-naphthyridine (7b) (150 mg,

0.527 mmol) in THF (3 mL) at 0 °C under nitrogen. After stirring at

room temperature for 3 h, the mixture was concentrated in vacuo, and

the residue was partitioned between CH₂Cl₂(20 mL) and water (20

mL). The layers were separated, and the organic phase was dried over

Na₂SO₄and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 100%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 3-((1-acetylpiperidin-

4-yl)methoxy)-4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)-

amino)piperidine-1-carboxylate (182 mg, 57%) as a yellow solid.

5.3 Hz, 1H), 6.39 (d, J = 6.5 Hz, 1H), 4.23 (br s, 1H), 3.88 (br s, 2H),

3.46−3.40 (m, 1H), 3.37−3.18 (m, 3H), 2.98−2.89 (m, 1H), 2.82 (br

s, 1H), 2.70 (br s, 2H), 2.62−2.53 (m, 1H), 2.47−2.39 (m, 1H), 2.16

(s, 3H), 2.05−1.97 (m, 1H), 1.90 (s, 3H), 1.71−1.59 (m, 1H), 1.58−

1.47 (m, 2H), 1.48−1.38 (m, 1H), 1.05−0.92 (m, 2H). One NH not

seen.

8-(((3R,4R)-3-(((trans)-4-Hydroxycyclohexyl)methoxy)-

piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-one

(26). Step 1: A solution of (3R,4R)-tert-butyl 4-azido-3-hydroxypiper-

idine-1-carboxylate (1 g, 4.1 mmol) in CH₂Cl₂(30 mL) at room

temperature was treated with imidazole (0.309 g, 4.54 mmol) and

TBDMSCl (0.684 g, 4.54 mmol), and the resulting mixture was stirred

at this temperature for 24 h. The organic phase was then washed with

water (30 mL), dried over MgSO₄, and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (25 g

column, gradient: 0 to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-

butyl 4-azido-3-((tert-butyldimethylsilyl)oxy)piperidine-1-carboxylate

(1.05 g, 71%) as a colorless oil. Step 2: (3R,4R)-tert-butyl 4-azido-3-

((tert-butyldimethylsilyl)oxy)piperidine-1-carboxylate (0.43 g, 1.2

mmol) was dissolved in EtOH (40 mL) and hydrogenated in the

H-Cube (ﬂow rate 1 mL/min, Pd/C cartridge, full H₂mode). The

solvent was then concentrated in vacuo. Puriﬁcation of the residue by

ﬂash chromatography on silica gel (25 g column, gradient: 0 to 10% (2

N NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-amino-3-

((tert-butyldimethylsilyl)oxy)piperidine-1-carboxylate (23) (0.38 g,

95%) as a colorless oil. Step 3: A ﬂask was charged with (3R,4R)-

tert-butyl 4-amino-3-((tert-butyldimethylsilyl)oxy) piperidine-1-car-

boxylate (23) (6.04 g, 18.3 mmol), 2-(benzyloxy)-8-chloro-3-methyl-

1,7-naphthyridine (7b) (4.0 g, 14 mmol), sodium tert-butoxide (4.05 g,

42.1 mmol), Brettphos (0.649 g, 1.21 mmol), and Brettphos

palladacycle (0.4 g, 0.5 mmol) and then ﬁlled with THF (10 mL).

The resulting mixture was stirred under nitrogen at room temperature

for 2 h. Pd₂(dba)₃(0.643 g, 0.702 mmol) and Brettphos (0.649 g, 1.21

mmol) were further added, and the mixture was stirred at 50 °C for 2

h, cooled to room temperature, and diluted with a saturated NH₄Cl

aqueous solution (20 mL). The aqueous phase was extracted with

EtOAc (40 mL). The organic phase was dried over MgSO₄and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (50 g column, gradient: 0 to 50%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-

methyl-1,7-naphthyridin-8-yl)amino)-3-((tert-butyldimethylsilyl)oxy)-

piperidine-1-carboxylate (8.05 g, 99%) as a yellow foam. LCMS (high-

1

pH method): t_R1.79 min, [M + H]⁺= 579.4. H NMR (400 MHz,

CDCl₃) δ ppm 7.90 (d, J = 5.9 Hz, 1H), 7.67 (d, J = 1.0 Hz, 1H), 7.50

(d, J = 7.1 Hz, 2H), 7.39−7.46 (m, 2H), 7.35 (s, 1H), 6.73 (d, J = 5.9

Hz, 1H), 6.16−6.26 (m, 1H), 5.51 (d, J = 16.9 Hz, 2H), 4.23−4.36

(m, 1H), 3.86−4.20 (m, 2H), 3.60−3.72 (m, 1H), 3.11−3.22 (m, 1H),

2.92−3.09 (m, 1H), 2.41 (d, J = 1.0 Hz, 3H), 2.21−2.31 (m, 1H), 1.45

(s, 9H), 0.77 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H). One NH not seen.

Step 4: A solution of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-

1,7-naphthyridin-8-yl)amino)-3-((tert-butyldimethylsilyl)oxy)-

piperidine-1-carboxylate (8.0 g, 14 mmol) in THF (50 mL) at room

temperature was treated with TBAF (1N in THF, 20 mL, 20 mmol),

and the resulting mixture was stirred at this temperature for 3 h and

then concentrated in vacuo. The residue was partitioned between

EtOAc (100 mL) and water (100 mL), and the layers were separated.

The organic phase was washed with water (2 × 100 mL) and brine

(100 mL), dried over MgSO₄, and concentrated in vacuo. Puriﬁcation

of the residue by ﬂash chromatography on silica gel (100 g column,

gradient: 0 to 100% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-

((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-hydroxypi-

peridine-1-carboxylate. The racemic mixture was separated by chiral

HLPC. The HPLC analysis was carried out on a Chiralcel OD-H (4.6

mm i.d. × 25 cm), using 25% EtOH in heptane at a ﬂow rate of 1 mL/

min. The material (5.3 g) was dissolved in a mixture of EtOH/heptane

(1:1) (250 mg in 1 mL). The puriﬁcation was carried out on a

Chiralcel OD (5 cm × 25 cm), using 25% EtOH in heptane at a ﬂow

rate of 50 mL/min, and 2 mL of solution was injected at a time. The

appropriate fractions were combined and concentrated in vacuo. The

residues were dissolved with CH₂Cl₂and concentrated under reduced

1

LCMS (high-pH method): t_R1.38 min, [M + H]⁺= 604.4. H NMR

(400 MHz, DMSO-d₆) δ ppm 7.86 (s, 1H), 7.82 (d, J = 5.5 Hz, 1H),

7.52 (d, J = 7.6 Hz, 2H),7.26−7.43 (m, 3H), 6.80 (d, J = 5.5 Hz, 1H),

6.50 (d, J = 7.6 Hz, 1H), 5.62 (d, J = 2.8 Hz, 2H), 4.20−4.33 (m, 1H),

3.78−3.96 (m, 3H), 3.67−3.76 (m, 1H), 3.42−3.54 (m, 2H), 3.33 (dd,

J = 9.3, 6.3 Hz, 1H), 3.14−3.26 (m, 2H), 3.08 (dd, J = 13.3, 8.1 Hz,

1H), 2.82 (br s, 2H), 2.65 (br s, 1H), 2.51 (d, J = 1.5 Hz, 1H), 2.37 (s,

3H), 2.13 (d, J = 4.0 Hz, 1H), 1.58−1.71 (m, 2H), 1.41−1.56 (m,

11H), 0.89−1.04 (m, 2H). Step 5: a solution of (3R,4R)-tert-butyl 3-

((1-acetylpiperidin-4-yl)methoxy)-4-((2-(benzyloxy)-3-methyl-1,7-

naphthyridin-8-yl)amino)piperidine-1-carboxylate (180 mg, 0.298

mmol) in TFA (2 mL) was heated at 50 °C for 2 h and then cooled

to room temperature and concentrated in vacuo. Puriﬁcation of the

residue by MDAP (high-pH method) gave 8-(((3R,4R)-3-((1-

acetylpiperidin-4-yl)methoxy)piperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (85 mg, 69%) as a yellow solid. LCMS (high-

1

pH method): t_R0.63 min, [M + H]⁺= 414. H NMR (400 MHz,

DMSO-d₆) δ ppm 7.74 (d, J = 5.3 Hz, 1H), 7.60 (s, 1H), 6.67 (d, J =

Q

DOI: 10.1021/acs.jmedchem.5b00773

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Journal of Medicinal Chemistry

Article

pressure to give the fastest running enantiomer, (3S,4S)-tert-butyl 4-

((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-hydroxypi-

peridine-1-carboxylate (2.4 g), and the slowest running enantiomer,

(3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)-

amino)-3-hydroxypiperidine-1-carboxylate (24) (2.2 g). LCMS (high-

pyridine (5.36 mL, 66.3 mmol) and, after 5 min, with the dropwise

addition of Tf₂O (11.2 mL, 66.3 mmol) over 10 min. The resulting

mixture was stirred at this temperature for 1 h and was then warmed to

room temperature. The organic phase was washed with water (2 × 50

mL) and then brine (50 mL), dried over MgSO₄, and concentrated in

vacuo to give (tetrahydro-2H-pyran-4-yl)methyl triﬂuoromethanesul-

fonate (14.2 g, 95%) as a colorless oil, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.38

(d, J = 6.6 Hz, 2H), 3.99−4.09 (m, 2H), 3.43 (td, J = 11.9, 2.0 Hz,

2H), 2.06−2.18 (m, 1H), 1.65−1.75 (m, 2H), 1.36−1.51 (m, 2H).

Step 2: a solution of (3R,4R)-tert-butyl 4-azido-3-hydroxypiperidine-1-

carboxylate (5) (10 g, 41 mmol) in DMF (50 mL) and THF (50 mL)

at 0 °C under nitrogen was treated portionwise with sodium hydride

(60% w/w in mineral oil, 2.2 g, 55 mmol). The resulting mixture was

stirred at this temperature for 30 min before being treated with the

dropwise addition of a solution of (tetrahydro-2H-pyran-4-yl)methyl

triﬂuoromethanesulfonate (13.3 g, 53.7 mmol) in THF (50 mL) over

20 min. The resulting mixture was then slowly allowed to warm to

room temperature over 2 h and then diluted with water (500 mL).

The aqueous phase was extracted with Et₂O (2 × 300 mL), and the

combined organics were washed with water (2 × 400 mL), dried over

MgSO₄, and concentrated in vacuo to give a yellow oil. Puriﬁcation of

this residue by ﬂash chromatography on silica gel (330 g column,

gradient: 0 to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-

azido-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate

(12.8 g, 91%) as a colorless oil, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ ppm

4.09−4.34 (m, 1H), 3.95−4.03 (m, 3H), 3.85−3.94 (m, 1H), 3.48−

3.57 (m, 1H), 3.34−3.47 (m, 4H), 3.12−3.22 (m, 1H), 2.85−2.96 (m,

1H), 2.61−2.82 (m, 1H), 1.77−1.99 (m, 2H), 1.61−1.73 (m, 2H),

1.47 (s, 9H), 1.29−1.43 (m, 2H). Step 3: a solution of (3R,4R)-tert-

butyl 4-azido-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-car-

boxylate (12.8 g, 37.6 mmol) in THF (100 mL) at room temperature

was treated with triphenylphosphine (10.8 g, 41.4 mmol), and the

resulting mixture was stirred at room temperature for 16 h. Water (30

mL) was then added, and the mixture was reﬂuxed for 24 h before

being cooled to room temperature and concentrated in vacuo. The

residue was dissolved in Et₂O (200 mL), and the solution was diluted

with cyclohexane (150 mL). Some solid separated after standing for 1

h at room temperature, and the solvent was partially evaporated,

causing further precipitation of triphenylphosphine oxide. The solid

was ﬁltered oﬀ, and the ﬁltrate was concentrated in vacuo to give a pale

yellow oil. Puriﬁcation of this residue by ﬂash chromatography on

silica gel (2 × 100 g column, gradient: 0 to 10% (2N NH₃in MeOH)

in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-amino-3-((tetrahydro-2H-pyran-

4-yl)methoxy)piperidine-1-carboxylate (8.7 g, 74%) as a pale yellow

1

pH method): t_R1.40 min, [M + H]⁺= 465.28. H NMR (400 MHz,

CDCl₃) δ ppm 7.77 (d, J = 5.9 Hz, 1H), 7.69 (d, J = 1.0 Hz, 1H), 7.50

(d, J = 7.1 Hz, 2H), 7.32−7.44 (m, 3H), 6.81 (d, J = 5.9 Hz, 1H), 6.30

(d, J = 4.2 Hz, 1H), 5.42−5.61 (m, 2H), 4.18−4.49 (m, 2H), 3.74−

3.87 (m, 1H), 3.50−3.61 (m, 1H), 2.63−2.90 (m, 2H), 2.42 (d, J = 0.7

Hz, 3H), 1.99−2.06 (m, 1H), 1.72−1.86 (m, 1H), 1.51 (s, 9H). One

H not seen. Step 5: 1,4-dioxaspiro[4.5]decan-8-ylmethyl triﬂuorome-

thanesulfonate was prepared from 1,4-dioxaspiro[4.5]decan-8-ylme-

thanol using the same procedure as that for the synthesis of (1-

acetylpiperidin-4-yl)methyl triﬂuoromethanesulfonate from 1-(4-

(hydroxymethyl)piperidin-1-yl)ethanone (see synthesis of 25, steps 1

and 2) (1.78 g, 100%, pale yellow oil). ¹H NMR (400 MHz, CDCl₃) δ

ppm 4.38 (d, J = 6.6 Hz, 2H), 3.86−4.05 (m, 4H), 1.76−1.95 (m, 5H),

1.59 (td, J = 13.4, 4.3 Hz, 2H), 1.32−1.48 (m, 2H). Step 6: a solution

of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-

yl)amino)-3-hydroxypiperidine-1-carboxylate (100 mg, 0.215 mmol;

see compounds 31 and 32, steps 1−4) at 0 °C under nitrogen in THF

(3 mL) was treated with sodium hydride (60% w/w in mineral oil,

21.5 mg, 0.538 mmol), and the resulting mixture was stirred for 20

min at this temperature before being treated with 1,4-dioxaspiro[4.5]-

decan-8-ylmethyl triﬂuoromethanesulfonate (131 mg, 0.431 mmol) as

a solution in THF (2 mL). After stirring at 0 °C for 2 h, the mixture

was diluted with water (10 mL) and extracted with EtOAc (2 × 10

mL). The combined organics were dried over Na₂SO₄and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (10 g column, gradient: 0 to 50%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 3-(1,4-dioxaspiro[4.5]-

decan-8-ylmethoxy)-4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-

yl)amino)piperidine-1-carboxylate (87 mg, 65%) as a colorless gum.

1

LCMS (high-pH method): t_R1.56 min, [M + H]⁺= 619.4. H NMR

(400 MHz, CDCl₃) δ ppm 7.90 (d, J = 5.6 Hz, 1H), 7.67 (d, J = 0.7

Hz, 1H), 7.51 (d, J = 7.1 Hz, 2H), 7.38−7.44 (m, 2H), 7.35 (d, J = 7.3

Hz, 1H), 6.75 (d, J = 5.9 Hz, 1H), 6.33−6.38 (m, 1H), 5.50 (d, J = 4.4

Hz, 2H), 4.18−4.31 (m, 1H), 3.79−3.92 (m, 5H), 3.43−3.55 (m, 2H),

3.31−3.38 (m, 2H), 3.19−3.28 (m, 2H), 2.40 (d, J = 0.7 Hz, 3H),

1.55−1.85 (m, 6H), 1.45 (s, 9H), 1.26−1.42 (m, 1H), 1.22−1.34 (m,

2H), 1.04−1.21 (m, 2H). Step 7: a solution of (3R,4R)-tert-butyl 3-

(1,4-dioxaspiro[4.5]decan-8-ylmethoxy)-4-((2-(benzyloxy)-3-methyl-

1,7-naphthyridin-8-yl)amino)piperidine-1-carboxylate (40 mg, 0.065

mmol) in CH₂Cl₂(5 mL) at room temperature was treated with TFA

(0.5 mL), and the resulting mixture was stirred at this temperature for

1 h and then concentrated in vacuo. The residue was dissolved in

MeOH (3 mL), cooled to 0 °C using an ice bath, and treated with

NaBH₄(12.2 mg, 0.323 mmol). After stirring for 2 h at this

temperature, the mixture was diluted with water (10 mL), and the

aqueous phase was extracted with EtOAc (2 × 10 mL). The combined

organic layers were dried over Na₂SO₄and concentrated in vacuo. The

residue was dissolved in MeOH (30 mL) and hydrogenated in the H-

cube on full hydrogen mode over a 10% Pd/C cartridge at 1 mL/min

ﬂow rate. The eluent was concentrated in vacuo, and the residue was

puriﬁed by MDAP (high-pH method) to give 8-(((3R,4R)-3-

(((1R,4R)-4-hydroxycyclohexyl) methoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (26) (4 mg, 16%). LCMS

1

oil. H NMR (400 MHz, CDCl₃) δ ppm 4.13−4.47 (m, 1H), 3.92−

4.09 (m, 3H), 3.49−3.60 (m, 1H), 3.40 (td, J = 11.7, 2.2 Hz, 2H), 3.31

(dd, J = 9.2, 6.5 Hz, 1H), 2.83−2.93 (m, 1H), 2.76 (br s, 2H), 2.35−

2.55 (m, 1H), 1.80−1.88 (m, 2H), 1.61−1.72 (m 2H), 1.52 (s, 2H),

1.47 (s, 9H), 1.23−1.43 (m, 3H). Step 4: a mixture of (3R,4R)-tert-

butyl 4-amino-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-

carboxylate (2.3 g, 7.3 mmol), 2-(benzyloxy)-8-chloro-3-methyl-1,7-

naphthyridine (7b) (1.5 g, 5.3 mmol), sodium 2-methylpropan-2-olate

(1.52 g, 15.8 mmol), Pd₂(dba)₃(0.241 g, 263 mmol), and BrettPhos

(0.283 g, 0.527 mmol) in THF (20 mL) was stirred at room

temperature under nitrogen for 1 h and was then stirred at 60 °C for 3

h before being cooled to room temperature and diluted with a

saturated NH₄Cl aqueous solution. The aqueous phase was extracted

with EtOAc (2 × 50 mL), and the combined organic phases were

dried over MgSO₄and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (50 g column, gradient: 0

to 70% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-

2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (2.72 g, 92%) as a

pale yellow foam. LCMS (high-pH method): t_R1.50 min, [M + H]⁺=

1

(high-pH method): t_R0.63 min, [M + H]⁺= 387. H NMR (600

MHz, DMSO-d₆) δ = 13.2 (br s, 1H), 7.73 (d, J = 5.1 Hz, 1H), 7.67 (s,

1H), 6.68 (d, J = 5.1 Hz, 1H), 6.59 (d, J = 7.7 Hz, 1H), 4.35 (d, J = 4.4

Hz, 1H), 4.17−4.08 (m, 1H), 3.34−3.28 (m, 1H) 3.22−3.13 (m, 4H),

2.89−2.83 (m, 1H), 2.49−2.44 (m, 1H), 2.32−2.26 (m, 1H), 2.12 (s,

3H), 1.99−1.93 (m, 1H), 1.68−1.62 (m, 2H), 1.55−1.45 (m, 2H),

1.34−1.19 (m, 2H), 1.00−0.91 (m, 2H), 0.78−0.69 (m, 2H). One NH

not seen.

3-Methyl-8-(((3R,4R)-3-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-one

(27). Step 1: a solution of (tetrahydro-2H-pyran-4-yl)methanol (7.0 g,

60 mmol) in CH₂Cl₂(50 mL) at 0 °C under nitrogen was treated with

1

563. H NMR (400 MHz, CDCl₃) δ ppm 7.90 (d, J = 5.9 Hz, 1H),

7.68 (d, J = 1.0 Hz, 1H), 7.49 (d, J = 7.1 Hz, 2H), 7.38−7.44 (m, 2H),

7.35 (d, J = 7.1 Hz, 1H), 6.75 (d, J = 5.9 Hz, 1H), 6.28−6.36 (m, 1H),

5.51 (s, 2H), 4.25−4.37 (m, 1H), 3.75−3.88 (m 2H), 3.43−3.52 (m,

R

DOI: 10.1021/acs.jmedchem.5b00773

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Journal of Medicinal Chemistry

Article

1H), 3.32 (d, J = 3.9 Hz, 1H), 3.12−3.27 (m, 3H), 2.89−3.11 (m,

1H), 2.41 (d, J = 0.7 Hz, 3H), 2.28−2.38 (m, 1H), 1.58−1.93 (m,

2H), 1.49−1.54 (m, 11H), 1.23−1.48 (m, 3H), 0.96−1.22 (m, 2H).

Step 5: a solution of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-

1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidine-1-carboxylate (0.18 g, 0.320 mmol) in MeOH (40

mL) was hydrogenated in the H-Cube on full mode using a 10% Pd/C

cartridge. The eluant was evaporated in vacuo to give a colorless glass.

This material was dissolved in CH₂Cl₂(10 mL), and the resulting

solution was treated with TFA (2 mL). The mixture was stirred at

room temperature for 1 h and then concentrated in vacuo. Puriﬁcation

of the residue by MDAP (high-pH method) gave 3-methyl-8-

(((3R,4R)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidin-4-yl)-

amino)-1,7-naphthyridin-2(1H)-one (27) (35 mg, 29%) as a yellow

dro-2H-pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-

2(1H)-one (29) (7 mg, 89%).

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-

2(1H)-one (30). Step 1: a solution of (tetrahydro-2H-thiopyran-4-

yl)methanol (10 g, 76 mmol) in MeOH (200 mL) at room

temperature was treated with a solution of sodium periodate (32.4

g, 151 mmol) in water (300 mL), and the resulting suspension was

stirred at 50 °C for 60 h, cooled to room temperature, and

concentrated in vacuo to leave a white, granular solid. This solid was

suspended in EtOAc (200 mL), stirred for 10 min, and then ﬁltered

oﬀ. The solid obtained was then suspended in 10% MeOH in CH₂Cl₂

(200 mL), stirred for 10 min, and then ﬁltered oﬀ. The combined

ﬁltrates were concentrated in vacuo to give 4-(hydroxymethyl)-

tetrahydro-2H-thiopyran 1,1-dioxide (11.2 g, 90%) as a colorless,

crystalline solid, which was used in the next step without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ ppm 3.59 (d, J = 6.4 Hz,

2H), 3.06−3.15 (m, 2H), 2.93−3.05 (m, 2H), 2.16−2.27 (m, 2H),

1.84−1.97 (m, 2H), 1.71−1.81 (m, 2H). Step 2: a solution of 4-

(hydroxymethyl)tetrahydro-2H-thiopyran 1,1-dioxide (3.0 g, 18.3

mmol) in CH₂Cl₂(20 mL) cooled at −10 °C using an acetone/ice

bath under nitrogen was treated with pyridine (1.62 mL, 20.1 mmol)

and then with Tf₂O (3.4 mL, 20.1 mmol) dropwise, and the resulting

mixture was stirred at this temperature for 1 h and then allowed to

warm to room temperature. The solution was then washed with brine

(10 mL), dried over MgSO₄, and concentrated in vacuo to give (1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methyl triﬂuoromethanesulfo-

nate (5.2 g, 96%) as a colorless solid, which was used in the next

step without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ ppm

4.42 (d, J = 6.1 Hz, 2H), 3.11−3.22 (m, 2H), 2.96−3.10 (m, 2H),

2.19−2.31 (m, 2H), 1.95−2.17 (m, 3H). Step 3: a solution of (3R,4R)-

tert-butyl 4-azido-3-hydroxypiperidine-1-carboxylate (5) (2.28 g, 9.41

mmol) in THF (10 mL) at 0 °C under nitrogen was treated with

potassium tert-butoxide (1 N in THF, 14.1 mL, 14.1 mmol), and the

resulting mixture was stirred at this temperature for 20 min. (1,1-

Dioxidotetrahydro-2H-thiopyran-4-yl)methyl triﬂuoromethanesulfo-

nate (4.18 g, 14.1 mmol) was then added in small portions over 10

min, and the resulting mixture was stirred at 0 °C for 2 h. Water (30

mL) was added, and the aqueous phase was extracted with EtOAc (2

× 20 mL). The combined organic phases were dried over MgSO₄and

concentrated in vacuo to give a pale yellow oil. Puriﬁcation of this

residue by ﬂash chromatography on silica gel (50 g column, gradient: 0

to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-azido-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

1

solid. LCMS (high-pH method): t_R0.67 min, [M + H]⁺= 373.2. H

NMR (400 MHz, CDCl₃) δ ppm 7.96 (d, J = 5.4 Hz, 1H), 7.66 (s,

1H), 6.68−6.74 (m, 1H), 6.29−6.49 (m, 1H), 4.37−4.51 (m, 1H),

3.73−3.87 (m, 2H), 3.43−3.50 (m, 2H), 3.33−3.42 (m, 2H), 3.18−

3.29 (m, 2H), 3.09−3.17 (m, 1H), 2.77−2.87 (m, 1H), 2.65−2.74 (m,

1H), 2.38 (s, 3H), 2.19−2.28 (m, 1H), 1.58 (m, 6H), 1.06−1.26 (m,

2H).

3-Methyl-8-(((3S,4S)-3-((tetrahydro-2H-pyran-4-yl)methoxy)-

piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-one (28) and 3-

Methyl-8-(((3R,4R)-3-((tetrahydro-2H-pyran-4-yl)methoxy)-

piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-one (29). Step 1:

a solution of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-

naphthyridin-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)-

piperidine-1-carboxylate (1.76 g, 3.13 mmol) in MeOH (100 mL) was

treated with 10% w/w palladium on charcoal (0.30 g, 0.282 mmol),

and the resulting mixture was stirred at room temperature under an

atmosphere of hydrogen (1 bar) for 3 h. The catalyst was ﬁltered oﬀ

using a 2.5 g pad of Celite and rinsed with MeOH. The combined

organics were concentrated in vacuo to give (3R,4R)-tert-butyl 4-((2-

hydroxy-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidine-1-carboxylate (1.50 g, 100%).

The racemic mixture was separated by chiral HLPC. The HPLC

analysis was carried out on a Chiralpak IC (4.6 mm i.d. × 25 cm),

using 20% EtOH (+0.2% isopropylamine) in heptane at a ﬂow rate of

1 mL/min. The material (40 mg) was dissolved in a mixture of EtOH/

heptane (1:1) (1 mL). The puriﬁcation was carried out on a Chiralpak

IC (30 mm × 25 cm), using 20% EtOH (+0.2% isopropylamine) in

heptane at a ﬂow rate of 30 mL/min, and 1 mL of solution was

injected. The appropriate fractions were combined and concentrated

under reduced pressure to give the fastest running enantiomer,

(3S,4S)-tert-butyl 4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-

yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-car-

boxylate (10 mg, 0.021 mmol), and the slowest running enantiomer,

(3R,4R)-tert-butyl 4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-

yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-car-

boxylate (10 mg, 0.021 mmol). LCMS (high-pH method): t_R1.47

min, [M + H]⁺= 473. ¹H NMR (400 MHz, CDCl₃) δ ppm 13.2 (br s,

1H), 7.96 (d, J = 5.4 Hz, 1H), 7.67 (d, J = 1.0 Hz, 1H), 6.71 (d, J = 5.6

Hz, 1H), 6.49−6.59 (m, 1H), 4.49−4.62 (m, 1H), 3.95−4.44 (m, 2H),

3.70−3.88 (m, 2H), 3.44−3.56 (m, 2H), 3.21 (d, J = 11.7 Hz, 4H),

2.32 (d, J = 0.7 Hz, 3H), 2.13−2.25 (m, 1H), 1.60−1.79 (m, 2H), 1.51

(s, 9H), 0.99−1.26 (m, 5H). Step 2: (3S,4S)-tert-butyl 4-((3-methyl-2-

oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidine-1-carboxylate (10 mg, 0.021 mmol)

and (3R,4R)-tert-butyl 4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyri-

din-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-

carboxylate (10 mg, 0.021 mmol) were dissolved in CH₂Cl₂and

treated with TFA (0.5 mL) at room temperature. The solutions were

stirred at this temperature for 2 h and then concentrated in vacuo. The

residues were dissolved in MeOH (2 mL) and loaded on to two 5 g

SCX-2 cartridges. The cartridges were washed with MeOH and then

eluted with a 2 N NH₃solution in MeOH. The ammoniac fractions

were concentrated in vacuo to give 3-methyl-8-(((3S,4S)-3-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-

2(1H)-one (28) (7 mg, 89%) and 3-methyl-8-(((3R,4R)-3-((tetrahy-

1

boxylate (3.32 g, 91%) as a pale yellow oil. H NMR (400 MHz,

CDCl₃) δ ppm 3.82−4.00 (m, 1H), 3.53−3.65 (m, 1H), 3.39−3.52

(m, 2H), 3.15−3.23 (m, 1H), 3.04−3.14 (m, 3H), 2.87−3.03 (m, 3H),

2.70−2.81 (m, 2H), 2.19 (d, J = 12.2 Hz, 2H), 1.79−2.01 (m, 4H),

1.38−1.51 (m, 9H). Step 4: a solution of (3R,4R)-tert-butyl 4-azido-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate (3.3 g, 8.5 mmol) in MeOH (100 mL) was treated with

palladium on charcoal (5% w/w, 50% wet, 1 g), and the resulting

mixture was stirred under an atmosphere of hydrogen (1 bar) for 2 h.

The catalyst was ﬁltered oﬀ using a pad of Celite (2.5 g), and the

ﬁltrate was concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (50 g column, gradient: 0 to 10% (2 N

NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-amino-3-((1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxy-

late (2.34 g, 76%) as a colorless crystalline solid. ¹H NMR (400 MHz,

CDCl₃) δ ppm 4.13−4.43 (m, 1H), 3.92−4.08 (m, 1H), 3.54−3.65

(m, 1H), 3.39 (dd, J = 9.2, 6.0 Hz, 1H), 3.05−3.14 (m, 2H), 2.87−

3.04 (m, 3H), 2.71−2.85 (m, 2H), 2.41−2.55 (m, 1H), 2.11−2.23 (m,

2H), 1.91−2.02 (m, 2H), 1.78−1.88 (m, 2H), 1.42−1.61 (m, 11H),

1.25−1.41 (m, 1H). Step 5: a mixture of (3R,4R)-tert-butyl 4-amino-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate (1.7 g, 4.7 mmol), 2-(benzyloxy)-8-chloro-3-methyl-1,7-

naphthyridine (7b) (1.10 g, 3.86 mmol), sodium 2-methylpropan-2-

olate (1.11 g, 11.6 mmol), BrettPhos (0.207 g, 0.386 mmol), and

Pd₂(dba)₃(0.177 g, 0.193 mmol) in THF (20 mL) was stirred under

nitrogen at room temperature for 1 h and was then stirred at 60 °C for

S

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3 h before being cooled to room temperature and diluted with a

saturated NH₄Cl aqueous solution. The aqueous phase was extracted

with EtOAc (2 × 50 mL), and the combined organic phases were

dried over MgSO₄and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (50 g column, gradient: 0

to 70% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (2.31 g,

98%) as a pale yellow foam. LCMS (high-pH method): t_R1.38 min,

[M + H]⁺= 611. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.90 (d, J = 5.6

Hz, 1H), 7.69 (s, 1H), 7.47−7.52 (m, 2H), 7.41 (t, J = 7.3 Hz, 2H),

7.35 (d, J = 7.3 Hz, 1H), 6.75 (d, J = 5.6 Hz, 1H), 6.22 (d, J = 8.6 Hz,

1H), 5.46−5.58 (m, 2H), 4.30−4.41 (m, 1H), 3.89−4.11 (m, 1H),

3.76−3.87 (m, 1H), 3.46−3.57 (m, 1H), 3.31 (d, J = 3.7 Hz, 4H), 2.67

(br s, 4H), 2.42 (s, 3H), 2.17−2.28 (m, 1H), 1.86−1.99 (m, 1H),

1.74−1.83 (m, 1H), 1.39−1.72 (m, 13H). Step 6: a solution of

(3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)-

amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidine-1-carboxylate (26 mg, 0.043 mmol) in TFA (2 mL) was

stirred at 50 °C for 2 h and then cooled to room temperature and

concentrated in vacuo. The residue was dissolved in MeOH and loaded

on to a 5 g SCX-2 cartridge. The cartridge was washed with MeOH

and then eluted with a 2 N NH₃solution in MeOH. The ammoniac

fractions were concentrated in vacuo. Puriﬁcation of the residue

obtained by MDAP (high-pH method) gave 8-(((3R,4R)-3-((1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidin-4-yl)amino)-

3-methyl-1,7-naphthyridin-2(1H)-one (30) (7.5 mg, 42%) as a beige

vacuo to give 8-(((3S,4S)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-

yl)methoxy)piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-2(1H)-

one (31) (26 mg, 94%) and 8-(((3R,4R)-3-((1,1-dioxidotetrahydro-

2H-thiopyran-4-yl)methoxy)piperidin-4-yl)amino)-3-methyl-1,7-naph-

thyridin-2(1H)-one (32) (12.4 mg, 45%) as beige solids.

8-(((3R,4R)-3-((1,1-Dioxidotetrahydrothiophen-3-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-

2(1H)-one (33). Step 1: (1,1-dioxidotetrahydrothiophen-3-yl)methyl

triﬂuoromethanesulfonate was prepared from 3-(hydroxymethyl)-

tetrahydrothiophene 1,1-dioxide using the same procedure as that

for the synthesis of (1-acetylpiperidin-4-yl)methyl triﬂuoromethane-

sulfonate from 1-(4-(hydroxymethyl)piperidin-1-yl)ethanone (1.72 g,

1

92%, pale yellow crystalline solid). H NMR (400 MHz, CDCl₃) δ

ppm 4.52−4.65 (m, 2H), 3.22−3.38 (m, 2H), 3.07−3.20 (m, 1H),

2.94−3.05 (m, 1H), 2.86−2.94 (m, 1H), 2.41−2.52 (m, 1H), 2.07 (dq,

J = 13.6, 9.4 Hz, 1H). Step 2: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-hydroxypiperi-

dine-1-carboxylate (24) (450 mg, 0.969 mmol) in THF (5 mL) under

nitrogen was cooled to 0 °C using an ice bath and was then treated

with potassium tert-butoxide (239 mg, 2.13 mmol). After stirring for

10 min, (1,1-dioxidotetrahydrothiophen-3-yl)methyl triﬂuorometha-

nesulfonate (547 mg, 1.94 mmol) was added, and the resulting mixture

was stirred for 2 h at this temperature. After standing over 2 days at

room temperature under a nitrogen atmosphere, the reaction mixture

was concentrated in vacuo. Puriﬁcation of the residue obtained by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 100%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-

methyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidotetrahydrothio-

phen-3-yl)methoxy)piperidine-1-carboxylate (55 mg, 10% yield) as a

pale yellow gum. LCMS (high-pH method): t_R1.38 min, [M + H]⁺=

1

solid. LCMS (high-pH method): t_R0.62 min, [M + H]⁺= 421.1. H

NMR (400 MHz, CDCl₃) δ ppm 13.2 (br s, 1H), 7.95 (d, J = 5.4 Hz,

1H), 7.67 (d, J = 1.0 Hz, 1H), 6.62−6.77 (m, 2H), 4.45−4.60 (m,

1H), 3.54−3.63 (m, 2H), 3.44 (dd, J = 8.9, 6.0 Hz, 2H), 3.24 (d, J =

12.7 Hz, 1H), 2.71−2.98 (m, 6H), 2.33−2.38 (m, 3H), 2.22−2.31 (m,

1H), 1.93−2.10 (m, 2H), 1.60−1.84 (m, 4H). One NH not seen.

8-(((3S,4S)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-1,7-naphthyridin-

2(1H)-one (31) and 8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-

thiopyran-4-yl)methoxy)piperidin-4-yl)amino)-3-methyl-1,7-

naphthyridin-2(1H)-one (32). Step 1: a solution of (3R,4R)-tert-

butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-hy-

droxypiperidine-1-carboxylate (75 mg, 0.16 mmol) (see steps 1−4 of

compound 26) and a solution of (3S,4S)-tert-butyl 4-((2-(benzyloxy)-

3-methyl-1,7-naphthyridin-8-yl)amino)-3-hydroxypiperidine-1-carbox-

ylate (75 mg, 0.16 mmol) in DMF (5 mL) at 0 °C under nitrogen

were treated with sodium hydride (60% w/w in mineral oil, 21.5 mg,

0.538 mmol), and the resulting mixtures were stirred at this

temperature for 10 min; then, (1,1-dioxidotetrahydro-2H-thiopyran-

4-yl)methyl triﬂuoromethanesulfonate (96 mg, 0.323 mmol) was

added as a solution in DMF (1 mL) to each reaction. The mixtures

were allowed to warm to room temperature over 2 and were then

diluted with water (10 mL) and extracted with EtOAc (10 mLl). In

each case, the organic layer was washed with water (10 mL), dried

over MgSO₄, and concentrated in vacuo. Puriﬁcation of each residue by

ﬂash chromatography on silica gel (10 g column, gradient: 0 to 100%

EtOAc in cyclohexane) gave (3S,4S)-tert-butyl 4-((2-(benzyloxy)-3-

methyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)methoxy)piperidine-1-carboxylate (40 mg, 41%) as a

colorless glass and (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-

naphthyridin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-

yl)methoxy)piperidine-1-carboxylate (37 mg, 37%) as a colorless glass.

Step 2: (3S,4S)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyr-

idin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidine-1-carboxylate (40 mg, 0.065 mmol) and (3R,4R)-

tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate (35 mg, 0.057 mmol) were each dissolved in TFA (3 mL),

and the solutions were stirred at 60 °C for 2 h, cooled to room

temperature, and concentrated in vacuo. The residues were dissolved in

MeOH (2 mL) and loaded on to two 5 g SCX-2 cartridges. The

cartridges were washed with MeOH and then eluted with a 2 N NH₃

solution in MeOH. The ammoniac fractions were concentrated in

1

597.4. H NMR (400 MHz, CDCl₃) δ ppm 7.89 (d, J = 5.6 Hz, 1H),

7.69 (s, 1H), 7.48 (d, J = 7.1 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.34 (d,

J = 7.3 Hz, 1H), 6.98 (dd, J = 17.1, 10.8 Hz, 1H), 6.77 (d, J = 5.9 Hz,

1H), 5.45−5.54 (m, 2H), 4.32−4.44 (m, 1H), 4.09−4.20 (m, 1H),

3.95−4.05 (m, 1H), 3.59−3.94 (m, 2H), 3.27−3.51 (m, 2H), 3.14−

3.26 (m, 2H), 2.99−3.12 (m, 1H), 2.75−2.97 (m, 2H), 2.25−2.43 (m,

4H), 2.14−2.24 (m, 1H), 1.93−2.12 (m, 1H), 1.68−1.84 (m, 1H),

1.51 (s, 9H), 1.23−1.37 (m, 1H). Step 3: a solution of (3R,4R)-tert-

butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-

((1,1-dioxidotetrahydrothiophen-3-yl)methoxy)piperidine-1-carboxy-

late (55 mg, 0.092 mmol) in TFA (1 mL) was reﬂuxed for 2 h, cooled

to room temperature, and concentrated in vacuo. The residue was

loaded on to a 5 g SCX cartridge, washed with MeOH, and eluted with

a 2 N NH₃solution in MeOH. The ammoniac fractions were

combined and concentrated in vacuo to give 8-(((3R,4R)-3-((1,1-

dioxidotetrahydrothiophen-3-yl)methoxy)piperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (33) (17 mg, 45%) as a beige

1

glass. LCMS (high-pH method): t_R0.61 min, [M + H]⁺= 407. H

NMR (400 MHz, CDCl₃) δ = 7.95 (d, J = 5.4 Hz, 1H), 7.67 (s, 1H),

6.72 (d, J = 5.4 Hz, 1H), 6.60 (d, J = 7.3 Hz, 1H), 4.55−4.43 (m, 1H),

3.79−3.67 (m, 1H), 3.67−3.59 (m, 1H), 3.58−3.52 (m, 1H), 3.44−

3.32 (m, 1H), 3.21−3.11 (m, 1H), 3.06−2.91 (m, 2H), 2.91−2.66 (m,

4H), 2.66−2.56 (m, 1H), 2.36 (s, 3H), 2.28−2.20 (m, 1H), 2.17−2.06

(m, 1H), 1.94−1.77 (m, 1H), 1.70 (d, J = 10.0 Hz, 1H). Two NH not

seen.

3-Methyl-8-(((3R,4R)-3-(2-(tetrahydro-2H-pyran-4-yl)-

ethoxy)piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-one (34).

Step 1: a solution of (3R,4R)-tert-butyl 4-azido-3-hydroxypiperidine-1-

carboxylate (5) (0.64 g, 2.64 mmol) in DMF (10 mL) under nitrogen

was cooled at 0 °C using an ice bath and then treated with sodium

hydride (60% w/w in mineral oil, 0.14 g, 3.50 mmol). The resulting

mixture was stirred at this temperature for 20 min; then, 4-(2-

bromoethyl)tetrahydropyran (0.61 g, 3.17 mmol) was added. After

stirring at 0 °C for 2 h, the reaction mixture was allowed to warm to

room temperature and stirred for a further 2 h. The reaction mixture

was then treated with water (30 mL), and the aqueous phase was

extracted with EtOAc (2 × 30 mL). The combined organics were

washed with water (2 × 30 mL), dried over Na₂SO₄, and concentrated

in vacuo. Puriﬁcation of this residue by ﬂash chromatography on silica

gel (25 g column, gradient: 0 to 50% EtOAc in cyclohexane) gave

T

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(3R,4R)-tert-butyl 4-azido-3-(2-(tetrahydro-2H-pyran-4-yl)ethoxy)-

piperidine-1-carboxylate (0.55 g, 59% yield) as a colorless oil. ¹H

NMR (400 MHz, CDCl₃) δ ppm 4.04−4.41 (m, 1H), 3.96 (s, 3H),

3.67−3.80 (m, 1H), 3.53−3.64 (m, 1H), 3.34−3.46 (m, 3H), 3.12−

3.22 (m, 1H), 2.81−2.92 (m, 1H), 2.70 (br s, 1H), 1.87−1.99 (m,

1H), 1.52−1.78 (m, 5H), 1.40−1.51 (m, 10H), 1.25−1.39 (m, 2H).

Step 2: a solution of (3R,4R)-tert-butyl 4-azido-3-(2-(tetrahydro-2H-

pyran-4-yl)ethoxy)piperidine-1-carboxylate (0.52 g, 1.467 mmol) in

MeOH (80 mL) was hydrogenated in the H-cube on full hydrogen

mode over a 10% Pd/C cartridge at 1 mL/min ﬂow rate. The eluant

was concentrated in vacuo to give (3R,4R)-tert-butyl 4-amino-3-(2-

(tetrahydro-2H-pyran-4-yl)ethoxy)piperidine-1-carboxylate (0.48 g,

100%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ ppm

4.13−4.55 (m, 1H), 4.00−4.09 (m, 1H), 3.96 (dd, J = 11.2, 3.9 Hz,

2H), 3.68−3.78 (m, 1H), 3.47−3.54 (m, 1H), 3.39 (t, J = 11.6 Hz,

2H), 2.84−2.93 (m, 1H), 2.68−2.80 (m, 2H), 2.35−2.56 (m, 1H),

1.79−1.87 (m, 1H), 1.52−1.74 (m, 6H), 1.45−1.50 (m, 10H), 1.25−

1.40 (m, 3H). Step 3: a suspension of 8-chloro-3-methyl-1,7-

naphthyridin-2(1H)-one (7a) (40 mg, 0.21 mmol), (3R,4R)-tert-

butyl 4-amino-3-(2-(tetrahydro-2H-pyran-4-yl)ethoxy)piperidine-1-

carboxylate (101 mg, 0.308 mmol) and Caddick catalyst (12 mg,

0.021 mmol) in THF (250 mL) at room temperature under nitrogen

was treated with LiHMDS (1 M in THF, 0.62 mL, 0.62 mmol) and

was then stirred at 60 °C for 3 h before being cooled to 0 °C and

quenched with a saturated NH₄Cl aqueous solution. The mixture was

diluted with EtOAc (30 mL), and the insoluble material was ﬁltered oﬀ

and then washed with EtOAc (2 × 50 mL) to give (3R,4R)-tert-butyl

4-((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-(2-

(tetrahydro-2H-pyran-4-yl)ethoxy)piperidine-1-carboxylate (52 mg,

52%) as a pale yellow solid. LCMS (high-pH method): t_R1.07 min,

[M + H]⁺= 487.4. Step 4: a solution of (3R,4R)-tert-butyl 4-((3-

methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-(2-(tetrahy-

dro-2H-pyran-4-yl)ethoxy)piperidine-1-carboxylate (52 mg, 0.107

mmol) in CH₂Cl₂(3 mL) at room temperature was treated with

TFA (1 mL). The resulting mixture was stirred at room temperature

for 1 h and was then concentrated in vacuo. The residue was loaded on

to a 5 g SCX cartridge, washed with MeOH (20 mL), and eluted with

a 2 N NH₃solution in MeOH (20 mL). The ammoniac fractions were

concentrated in vacuo to give 3-methyl-8-(((3R,4R)-3-(2-(tetrahydro-

2H-pyran-4-yl)ethoxy)piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-

one (34) (35 mg, 85% yield) as a brown solid. LCMS (high-pH

method): t_R0.68 min, [M + H]⁺= 387. ¹H NMR (400 MHz, CDCl₃)

δ ppm 0.98−1.18 (m, 2H), 1.27−1.75 (m, 7H), 2.14−2.25 (m, 1H),

2.36 (s, 3H), 2.63−2.73 (m, 1H), 2.80 (br t, J = 10.3 Hz, 1H), 2.94 (br

t, J = 12.0 Hz, 1H), 3.04−3.13 (m, 1H), 3.09−3.18 (m, 1H), 3.37−

3.47 (m, 2H), 3.51−3.60 (m, 1H), 3.63−3.71 (m, 2H), 3.75 (br dd, J

= 11.4, 3.7 Hz, 1H), 4.37−4.52 (m, 1H), 6.41 (br s, 1H), 6.69 (d, J =

5.5 Hz, 1H), 7.65 (q, J = 1.5 Hz, 1H), 7.94 (d, J = 5.4 Hz, 1H), 13.09

(br s, 1H).

4H), 0.96−1.25 (m, 2H). Step 2: a 20 mL microwave vial was charged

with K₂CO₃(172 mg, 1.25 mmol), Pd(OAc)₂(7.0 mg, 0.031 mmol),

3-pyridineboronic acid pinacol ester (128 mg, 0.623 mmol), (3R,4R)-

tert-butyl 4-((2-(benzyloxy)-5-bromo-3-methyl-1,7-naphthyridin-8-yl)-

amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxy-

late (200 mg, 0.312 mmol), and di((3S,5S,7S)-adamantan-1-yl)-

(butyl)phosphine (cataCXium A) (11.18 mg, 0.031 mmol) and was

then ﬁlled with 1,4-dioxane (8 mL) and water (4 mL). The reaction

mixture was degassed for 20 min with nitrogen and then stirred under

microwave irradiation at 100 °C for 1 h before being cooled to room

temperature. The solvent was removed in vacuo, and the residue was

partitioned between CH₂Cl₂(20 mL) and water (20 mL). The layers

were separated, and the organic phase was dried using a hydrophobic

frit and then concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (10 g column, gradient: 0 to 8% MeOH

in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-5-

(pyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-pyran-

4-yl)methoxy)piperidine-1-carboxylate (133 mg, 60%). LCMS (high-

1

pH method): t_R1.45 min, [M + H]⁺= 640. H NMR (400 MHz,

DMSO-d₆) δ ppm 8.59−8.66 (m, 2H), 7.84−7.88 (m, 1H), 7.82 (d, J

= 1.2 Hz, 1H), 7.80 (s, 1H), 7.50−7.58 (m, 3H), 7.37−7.43 (m, 2H),

7.33 (s, 1H), 6.93−6.98 (m, 1H), 5.59−5.73 (m, 2H), 4.26−4.36 (m,

1H), 3.76−3.87 (m, 1H), 3.58−3.66 (m, 2H), 3.45−3.54 (m, 1H),

3.38−3.44 (m, 1H), 3.19−3.26 (m, 1H), 3.06 (br s, 3H), 2.31 (d, J =

0.7 Hz, 3H), 2.10−2.23 (m, 1H), 2.01−2.09 (m, 1H), 1.88−1.98 (m,

1H), 1.53−1.77 (m, 2H), 1.44 (s, 9H), 1.26−1.36 (m, 2H), 0.83−1.04

(m, 2H). Step 3: a ﬂask was charged with (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-5-(pyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-

3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (102

mg, 0.159 mmol) and then ﬁlled with TFA (2 mL), and the resulting

mixture was stirred at 80 °C for 2 h, cooled to room temperature, and

concentrated in vacuo. Puriﬁcation of the residue by MDAP (high-pH

method) gave 3-methyl-5-(pyridin-3-yl)-8-(((3R,4R)-3-((tetrahydro-

2H-pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-

2(1H)-one (34 mg, 47%) as a colorless foam. LCMS (high-pH

method): t_R0.66 min, [M + H]⁺= 450. ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 11.50 (br s, 1H), 8.62 (dd, J = 4.8, 1.6 Hz, 1H), 8.59 (d, J =

1.7 Hz, 1H), 7.80 (s, 1H), 7.72 (s, 1H), 7.50−7.55 (m, 2H), 6.84−6.88

(m, 1H), 4.13−4.27 (m, 1H), 3.62−3.72 (m, 2H), 3.37−3.43 (m, 1H),

3.07−3.29 (m, 5H), 2.84−2.93 (m, 1H), 2.26−2.36 (m, 1H), 2.09 (d, J

= 1.2 Hz, 3H), 1.96−2.05 (m, 1H), 1.56−1.69 (m, 1H), 1.32−1.41 (m,

3H), 0.93−1.10 (m, 2H). Two NH not seen.

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-5-(pyridin-3-yl)-1,7-

naphthyridin-2(1H)-one (36). Step 1: a solution of (3R,4R)-tert-

butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate (2.6 g, 4.3 mmol) in CH₂Cl₂(50 mL) at −10 °C was treated

with NBS (0.66 g, 3.71 mmol), and the resulting mixture stirred at this

temperature for 30 min before being treated with a saturated sodium

metabisulphite aqueous solution (50 mL). The resulting mixture was

stirred for 20 min at room temperature, and the layers were separated.

The organic phase was dried over MgSO₄and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (50 g

column, gradient: 0 to 50% EtOAc in cyclohexane) gave (3R,4R)-tert-

butyl 4-((2-(benzyloxy)-5-bromo-3-methyl-1,7-naphthyridin-8-yl)-

amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidine-1-carboxylate (2.71 g, 92%) as an orange solid. LCMS

3-Methyl-5-(pyridin-3-yl)-8-(((3R,4R)-3-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-

2(1H)-one (35). Step 1: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-

2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (2.7 g, 4.8 mmol) in

CH₂Cl₂at 0 °C was treated with NBS (0.683 g, 3.84 mmol), and the

resulting mixture was stirred at this temperature for 30 min. Further

NBS (50 mg, 0.28 mmol) was added, and the mixture was stirred for

another 10 min, warmed to room temperature, and washed with a

saturated sodium metabisulphite aqueous solution. The organic phase

was then dried over MgSO₄and concentrated in vacuo to give

(3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-methyl-1,7-naphthyr-

idin-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-

carboxylate (2.92 g, 95%) as a beige foam, which was used in the next

step without further puriﬁcation. LCMS (high-pH method): t_R1.68

1

(high-pH method): t_R1.54 min, [M + H]⁺= 688/690 (1 Br). H

NMR (400 MHz, CDCl₃) δ ppm 8.03 (s, 1H), 8.00 (d, J = 1.2 Hz,

1H), 7.46−7.50 (m, 2H), 7.38−7.44 (m, 2H), 7.36 (d, J = 7.3 Hz,

1H), 6.21 (d, J = 8.3 Hz, 1H), 5.47−5.57 (m, 2H), 4.24−4.35 (m,

1H), 3.85−4.10 (m, 1H), 3.76−3.84 (m, 1H), 3.47−3.56 (m, 1H),

3.03−3.34 (m, 3H), 2.88−2.97 (m, 1H), 2.65−2.86 (m, 3H), 2.48 (d, J

= 0.7 Hz, 3H), 2.17−2.26 (m, 1H), 1.89−2.01 (m, 1H), 1.77−1.86 (m,

1H), 1.64−1.76 (m, 1H), 1.52 (s, 9H), 1.45−1.65 (m, 4H). Step 2: a

20 mL microwave vial was charged with pyridin-3-ylboronic acid (21.4

mg, 0.174 mmol), (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-

methyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)methoxy)piperidine-1-carboxylate (80 mg, 0.12 mmol),

1

min, [M + H]⁺= 641/643 (1 Br). H NMR (400 MHz, CDCl₃) δ

ppm 8.03 (s, 1H), 7.99 (d, J = 1.2 Hz, 1H), 7.46−7.50 (m, 2H), 7.38−

7.44 (m, 2H), 7.36 (d, J = 7.1 Hz, 1H), 6.29 (d, J = 7.8 Hz, 1H), 5.52

(s, 2H), 4.17−4.30 (m, 1H), 3.82 (d, J = 7.6 Hz, 3H), 3.43−3.52 (m,

1H), 3.30 (d, J = 3.9 Hz, 1H), 3.14−3.25 (m, 3H), 2.46 (d, J = 1.0 Hz,

2H), 2.26−2.36 (m, 1H),1.60−1.72 (m, 2H), 1.52 (s, 11H), 1.50 (br s,

U

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

K₂CO₃(64.1 mg, 0.464 mmol), Pd(OAc)₂(2.6 mg, 0.012 mmol), and

di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cataCXium A) (4.2

mg, 0.012 mmol) and then ﬁlled with 1,4-dioxane (4 mL) and water (2

mL). The resulting mixture was degassed under vacuum, quenched

with nitrogen for 20 min, and stirred under nitrogen and microwave

irradiation at 100 °C for 30 min before being cooled to room

temperature and concentrated in vacuo. The residue was partitioned

between CH₂Cl₂(20 mL) and water (20 mL), and the layers were

separated. The organic layer was dried using a hydrophobic frit and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 10% (2

N NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 3-((1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-4-((3-methyl-2-oxo-5-

(pyridin-3-yl)-1,2-dihydro-1,7-naphthyridin-8-yl)amino)piperidine-1-

carboxylate. This derivative was dissolved in TFA (2 mL), and the

resulting solution was stirred at reﬂux for 16 h, cooled to room

temperature, and concentrated in vacuo. The residue was loaded on a 5

g SCX cartridge, washed with MeOH, and eluted with a 2 N NH₃

solution in MeOH. The ammoniac fractions were concentrated in

vacuo to give 8-(((3R,4R)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-

yl)methoxy)piperidin-4-yl)amino)-3-methyl-5-(pyridin-3-yl)-1,7-naph-

thyridin-2(1H)-one (36) (32 mg, 55%) as yellow solid. LCMS (high-

separated by chiral HLPC. The HPLC analysis was carried out on a

Chiralpak IC (4.6 mm i.d. × 25 cm), using 40% EtOH in heptane at a

ﬂow rate of 1 mL/min. The material (120 mg) was dissolved in a

mixture of EtOH/heptane (2:1) (1 mL). The puriﬁcation was carried

out on a Chiralpak IC (30 mm × 25 cm), using 40% EtOH in heptane

at a ﬂow rate of 30 mL/min, and 1 mL of solution was injected at a

time. The appropriate fractions were combined and concentrated in

vacuo to give the fastest running enantiomer, (3S,4S)-tert-butyl 4-((3-

methyl-5-(5-methylpyridin-3-yl)-2-oxo-1,2-dihydro-1,7-naphthyridin-

8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-car-

boxylate (58 mg), and the slowest running enantiomer, (3R,4R)-tert-

butyl 4-((3-methyl-5-(5-methylpyridin-3-yl)-2-oxo-1,2-dihydro-1,7-

naphthyridin-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)-

piperidine-1-carboxylate (53 mg). Step 3: a solution of (3R,4R)-tert-

butyl 4-((3-methyl-5-(5-methylpyridin-3-yl)-2-oxo-1,2-dihydro-1,7-

naphthyridin-8-yl)amino)-3-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-

piperidine-1-carboxylate (58 mg, 0.10 mmol) in CH₂Cl₂(3 mL) at

room temperature was treated with TFA (1 mL), and the resulting

mixture was stirred at this temperature for 30 min and then

concentrated in vacuo. The residue was loaded on a 2 g SCX-2

cartridge, washed with MeOH, and then eluted with a 2 N NH₃

solution in MeOH. The ammoniac fractions were combined and

concentrated in vacuo to give 3-methyl-5-(5-methylpyridin-3-yl)-8-

(((3R,4R)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidin-4-yl)-

amino)-1,7-naphthyridin-2(1H)-one (37) (43 mg, 90%). LCMS

1

pH method): t_R0.61 min, [M + H]⁺= 498. H NMR (400 MHz,

DMSO-d₆) δ ppm 11.50 (br s, 1H), 8.55−8.68 (m, 1H), 7.83 (d, J =

7.8 Hz, 1H), 7.73 (s, 1H), 7.47−7.58 (m, 2H), 6.85 (d, J = 7.6 Hz,

1H), 4.19 (br s, 1H), 3.46 (dd, J = 9.3, 6.4 Hz, 1H), 3.21−3.28 (m,

2H), 3.17 (d, J = 2.4 Hz, 1H), 2.94−3.05 (m, 2H), 2.82−2.93 (m,

3H), 2.29−2.41 (m, 1H), 2.09 (s, 3H), 1.99−2.06 (m, 1H), 1.82−1.96

(m, 2H), 1.67−1.79 (m, 1H), 1.47−1.49 (m, 1H), 1.41−1.55 (m, 3H),

1.35 (d, J = 9.3 Hz, 2H).

1

(method TFA): t_R0.48 min, [M + H]⁺= 464. H NMR (400 MHz,

CDCl₃) δ ppm 13.2 (br s, 1H), 8.52 (d, J = 1.5 Hz, 1H), 8.47 (d, J =

1.7 Hz, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.53 (s, 1H), 6.69 (d, J = 6.6

Hz, 1H), 4.51 (d, J = 4.4 Hz, 1H), 3.76−3.89 (m, 2H), 3.39 (dd, J =

9.3, 6.6 Hz, 2H), 3.26 (qd, J = 11.8, 2.0 Hz, 2H), 3.10−3.18 (m, 1H),

2.83 (t, J = 10.1 Hz, 1H), 2.70 (dd, J = 12.2, 8.8 Hz, 1H), 2.47 (s, 3H),

2.35 (m, 4H), 2.22−2.30 (m, 1H), 1.64−1.84 (m, 3H), 1.41−1.56 (m,

2H), 1.07−1.29 (m, 2H). One NH not seen.

3-Methyl-5-(5-methylpyridin-3-yl)-8-(((3R,4R)-3-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naph-

thyridin-2(1H)-one (37). Step 1: a solution of (3R,4R)-tert-butyl 4-

((3-methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-((tetra-

hydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (1.5 g, 3.2

mmol) in CHCl₃(20 mL) at −10 °C was treated with NBS (0.508

g, 2.86 mmol), and the resulting mixture was stirred at this

temperature for 1 h and then treated with a saturated sodium

metabisulphite aqueous solution (20 mL). The biphasic mixture was

warmed to room temperature and stirred for 10 min, and the layers

were separated. The organic phase was dried using a phase separator

and concentrated in vacuo to give (3R,4R)-tert-butyl 4-((5-bromo-3-

methyl-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)amino)-3-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (1.63 g, 93%) as

a yellow solid, which was used in the next step without further

puriﬁcation. LCMS (high-pH method): t_R1.22 min, [M + H]⁺= 551/

553 (1 Br). ¹H NMR (400 MHz, CDCl₃) δ ppm 13.21 (br s, 1H), 8.08

(s, 1H), 8.04 (s, 1H), 6.58 (d, J = 7.1 Hz, 1H), 4.41−4.54 (m, 1H),

4.15−4.35 (m, 2H), 3.96−4.06 (m, 1H), 3.82 (br s, 2H), 3.41−3.56

(m, 2H), 3.03−3.35 (m, 4H), 2.36 (s, 3H), 2.15−2.25 (m, 1H), 1.67

(br s, 2H), 1.52 (s, 9H), 1.33−1.48 (m, 2H), 1.03−1.25 (m, 2H). Step

2: a 20 mL microwave vial was charged with (5-methylpyridin-3-

yl)boronic acid (64.6 mg, 0.471 mmol), (3R,4R)-tert-butyl 4-((5-

bromo-2-hydroxy-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (200 mg, 0.363

mmol), K₂CO₃(150 mg, 1.09 mmol), Pd(OAc)₂(8.1 mg, 0.036

mmol), and di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cata-

CXium A) (13 mg, 0.036 mmol) and then ﬁlled with 1,4-dioxane (4

mL) and water (2 mL). The resulting mixture was degassed for 20 min

under vacuum, quenched with nitrogen, and stirred at 100 °C for 30

min under microwave irradiation before being cooled to room

temperature and concentrated in vacuo. The residue was partitioned

between CH₂Cl₂(20 mL) and water (20 mL), and the layers were

separated. The organic phase was dried using a hydrophobic frit and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, gradient: 0 to 10% (2N

NH₃in MeOH) in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-((3-methyl-5-

(5-methylpyridin-3-yl)-2-oxo-1,2-dihydro-1,7-naphthyridin-8-yl)-

amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxy-

late (0.178 g, 87%) as a pale yellow solid. The racemic mixture was

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-

3-yl)-1,7-naphthyridin-2(1H)-one (38). A 20 mL microwave vial

was charged with (5-methylpyridin-3-yl)boronic acid (34.2 mg, 0.249

mmol), (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-methyl-1,7-

naphthyridin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-

yl)methoxy)piperidine-1-carboxylate (see preparation of 36) (86 mg,

0.12 mmol), K₂CO₃(68.9 mg, 0.499 mmol), Pd(OAc)₂(2.8 mg, 0.012

mmol), and di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cata-

CXium A) (4.5 mg, 0.012 mmol) and then ﬁlled with 1,4-dioxane

(4 mL) and water (2 mL). The reaction mixture was stirred and

degassed for 20 min with nitrogen and was then stirred at 100 °C for

30 min under microwave irradiation before being cooled to room

temperature and concentrated in vacuo. The residue was partitioned

between CH₂Cl₂(20 mL) and water (20 mL), and the layers were

separated. The organic phase was dried through a hydrophobic frit and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (10 g column, gradient: 0 to 10% (2N

NH₃in MeOH) in CH₂Cl₂gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-

3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate as a pale yellow solid. This residue was dissolved in TFA (3

mL), and the resulting solution was reﬂuxed for 2 h before being

cooled to room temperature and concentrated in vacuo. Puriﬁcation of

the residue by MDAP (high-pH method) gave 8-(((3R,4R)-3-((1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidin-4-yl)amino)-

3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one (38)

(47 mg, 74%) as a pale yellow solid. LCMS (method TFA): t_R0.68

min, [M + H]⁺= 512.3. ¹H NMR (400 MHz, CD₃OD) δ ppm 8.77−

8.80 (m, 1H), 8.73−8.76 (m, 1H), 8.40−8.46 (m, 1H), 7.82−7.86 (m,

1H), 7.77−7.80 (m, 1H), 4.44−4.55 (m, 1H), 3.87−3.95 (m, 1H),

3.64−3.76 (m, 2H), 3.46−3.59 (m, 2H), 3.21−3.30 (m, 2H), 3.11−

3.19 (m, 1H), 2.91−3.11 (m, 2H), 2.79−2.89 (m, 1H), 2.64 (s, 3H),

2.39−2.50 (m, 1H), 2.30 (s, 3H), 2.09−2.21 (m, 1H), 2.01−2.09 (m,

1H), 1.91−2.00 (m, 1H), 1.73−1.90 (m, 2H), 1.56−1.71 (m, 1H).

Three NH protons not seen.

V

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

5-(5-Methoxypyridin-3-yl)-3-methyl-8-(((3R,4R)-3-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naph-

thyridin-2(1H)-one (39). Step 1: a 20 mL microwave vial was

charged with K₂CO₃(172 mg, 1.25 mmol), Pd(OAc)₂(7.0 mg, 0.031

mmol), 3-methoxypyridine-5-boronic acid pinacol ester (147 mg,

0.623 mmol), (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-methyl-

1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidine-1-carboxylate (200 mg, 0.312 mmol), and di-

((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cataCXium A) (11.2

mg, 0.031 mmol) and then ﬁlled with 1,4-dioxane (8 mL) and water (4

mL). The resulting mixture was degassed under vacuum, quenched

with nitrogen over 20 min, and stirred under microwave irradiation at

100 °C for 1 h before being cooled to room temperature and

concentrated in vacuo. The residue was partitioned between CH₂Cl₂

(20 mL) and water (20 mL), and the layers were separated. The

organic phase was dried using a hydrophobic frit and then

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (10 g column, gradient: 2 to 8%

MeOH in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-(5-

methoxypyridin-3-yl)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tet-

rahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (120 mg,

46%) as a pale yellow solid. LCMS (method TFA): t_R1.47 min, [M

+ H]⁺= 670. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.33 (d, J = 2.9

Hz, 1H), 8.22 (d, J = 1.7 Hz, 1H), 7.87 (d, J = 1.0 Hz, 1H), 7.82 (s,

1H), 7.54 (d, J = 7.1 Hz, 2H), 7.37−7.44 (m, 3H), 7.30−7.36 (m,

1H), 6.92−6.99 (m, 1H), 5.59−5.74 (m, 2H), 4.25−4.36 (m, 1H),

3.90−4.15 (m, 2H), 3.89 (s, 3H), 3.76−3.85 (m, 1H), 3.57−3.66 (m,

2H), 3.45−3.54 (m, 1H), 3.37−3.44 (m, 1H), 3.20−3.26 (m, 1H),

3.07 (m, 2H), 2.70−3.12 (m, 1H), 2.32 (d, J = 0.7 Hz, 3H), 2.00−2.09

(m, 1H), 1.52−1.66 (m, 2H), 1.44 (s, 9H), 1.26−1.37 (m, 2H), 0.88−

1.05 (m, 2H). Step 2: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-5-(5-methoxypyridin-3-yl)-3-methyl-1,7-naphthyridin-8-

yl)amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-car-

boxylate (130 mg, 0.194 mmol) in TFA (2 mL) was stirred at 80 °C

for 2 h, cooled to room temperature, and concentrated in vacuo.

Puriﬁcation of the residue by MDAP (high-pH method) gave 5-(5-

methoxypyridin-3-yl)-3-methyl-8-(((3R,4R)-3-((tetrahydro-2H-pyran-

4-yl)methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-one

(39) (66 mg, 71%) as a colorless foam. LCMS (high-pH method): t_R

temperature, and concentrated in vacuo. The residue was loaded on a 5

g SCX cartridge, washed with MeOH, and eluted with a 2 N NH₃

solution in MeOH. The ammoniac fractions were concentrated in

vacuo to give 8-(((3R,4R)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-

yl)methoxy)piperidin-4-yl)amino)-5-(5-methoxypyridin-3-yl)-3-meth-

yl-1,7-naphthyridin-2(1H)-one (28 mg, 46%) as yellow solid. LCMS

1

(high-pH method): t_R0.70 min, [M + H]⁺= 528. H NMR (400

MHz, CDCl₃) δ ppm 13.20 (br s, 1H), 8.39 (d, J = 2.7 Hz, 1H), 8.27

(s, 1H), 7.92 (s, 1H), 7.77 (s, 1H), 7.26 (d, J = 1.5 Hz, 1H), 6.75 (d, J

= 7.3 Hz, 1H), 4.76 (br s, 1H), 4.51 (d, J = 3.7 Hz, 1H), 3.95 (s, 3H),

3.51−3.62 (m, 2H), 3.48 (dd, J = 8.7, 6.2 Hz, 1H), 3.38−3.44 (m,

1H), 3.16 (d, J = 12.5 Hz, 1H), 2.75−2.99 (m, 5H), 2.69 (dd, J = 11.7,

9.0 Hz, 1H), 2.34 (s, 3H), 2.25 (d, J = 10.0 Hz, 1H), 2.05 (t, J = 12.3

Hz, 2H), 1.62−1.90 (m, 4H).

3-Methyl-5-(pyrimidin-5-yl)-8-(((3R,4R)-3-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-

2(1H)-one (41). Step 1: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-

2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (2.7 g, 4.8 mmol) in

CH₂Cl₂at 0 °C was treated with NBS (0.683 g, 3.84 mmol), and the

resulting solution was stirred at this temperature for 30 min. Further

NBS (50 mg, 0.28 mmol) was added. The solution was stirred at 0 °C

for 10 min before being treated with a saturated sodium

metabisulphite aqueous solution. The layers were separated, and the

organic phase was dried using a phase separator and concentrated in

vacuo to give (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-methyl-

1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidine-1-carboxylate (2.92 g, 95%) as a beige foam,

which was used in the next step without further puriﬁcation. LCMS

1

(high-pH method): t_R1.67 min, [M + H]⁺= 643.5 (1 Br). H NMR

(400 MHz, CDCl₃) δ ppm 8.03 (s, 1H), 7.99 (d, J = 1.2 Hz, 1H),

7.46−7.50 (m, 2H), 7.38−7.44 (m, 2H), 7.36 (d, J = 7.1 Hz, 1H), 6.29

(d, J = 7.8 Hz, 1H), 5.52 (s, 2H), 4.14−4.31 (m, 1H), 3.82 (d, J = 7.6

Hz, 2H), 3.41−3.54 (m, 1H), 3.30 (d, J = 3.9 Hz, 1H), 2.92−3.25 (m,

4H), 2.46 (d, J = 1.0 Hz, 2H), 2.26−2.36 (m, 1H), 1.61−1.71 (m,

2H), 1.33−1.55 (m, 15H), 1.11−1.24 (m, 1H), 0.98−1.10 (m, 1H).

Step 2: a 20 mL microwave vial was charged with K₂CO₃(172 mg,

1.25 mmol), Pd(OAc)₂(7.00 mg, 0.031 mmol), pyrimidine-5-boronic

acid (77 mg, 0.62 mmol), (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-

bromo-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidine-1-carboxylate (200 mg, 0.312 mmol),

and di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cataCXium A)

(11 mg, 0.031 mmol) and then ﬁlled with 1,4-dioxane (8 mL) and

water (4 mL). The resulting mixture was stirred, degassed for 20 min

with nitrogen, and stirred at 100 °C under microwave irradiation for 1

h before being cooled to room temperature and concentrated in vacuo.

The residue was partitioned between CH₂Cl₂(20 mL) and water (20

mL), and the layers were separated. The organic phase was dried using

a hydrophobic frit and then concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (25 g column, gradient: 2

to 8% MeOH in CH₂Cl₂) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-

methyl-5-(pyrimidin-5-yl)-1,7-naphthyridin-8-yl)amino)-3-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (120 mg, 60%).

1

0.70 min, [M + H]⁺= 480. H NMR (400 MHz, DMSO-d₆) δ ppm

11.50 (br s, 1H), 8.32−8.36 (m, 1H), 8.13−8.20 (m, 1H), 7.72−7.78

(m, 1H), 7.58 (d, J = 1.2 Hz, 1H), 7.37 (dd, J = 2.7, 2.0 Hz, 1H), 6.85

(d, J = 7.6 Hz, 1H), 4.13−4.27 (m, 1H), 3.89 (s, 3H), 3.63−3.73 (m,

2H), 3.40 (dd, J = 9.3, 6.6 Hz, 1H), 3.18−3.29 (m, 3H), 3.14 (tt, J =

11.7, 2.4 Hz, 2H), 2.89 (d, J = 12.5 Hz, 1H), 2.26−2.37 (m, 1H), 2.10

(d, J = 1.0 Hz, 3H), 2.00 (d, J = 8.8 Hz, 1H), 1.63 (br s, 2H), 1.28−

1.44 (m, 3H), 0.95−1.10 (m, 2H). One NH not seen.

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-5-(5-methoxypyridin-3-yl)-3-

methyl-1,7-naphthyridin-2(1H)-one (40). Step 1: a 20 mL

microwave vial was charged with (5-methoxypyridin-3-yl)boronic

acid (26.6 mg, 0.174 mmol), (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-

bromo-3-methyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidotetrahy-

dro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (see prepara-

tion of 36) (80 mg, 0.116 mmol), K₂CO₃(64.1 mg, 0.464 mmol),

Pd(OAc)₂(2.60 mg, 0.012 mmol), and di((3S,5S,7S)-adamantan-1-

yl)(butyl)phosphine (cataCXium A) (4.2 mg, 0.012 mmol) and then

ﬁlled with 1,4-dioxane (4 mL) and water (2 mL). The resulting

mixture was degassed under vacuum, quenched with nitrogen for 20

min, and stirred under nitrogen and microwave irradiation at 100 °C

for 30 min before being cooled to room temperature and concentrated

in vacuo. The residue was partitioned between CH₂Cl₂(20 mL) and

water (20 mL), and the layers were separated. The organic layer was

dried using a hydrophobic frit and concentrated in vacuo. Puriﬁcation

of the residue by ﬂash chromatography on silica gel (25 g column,

gradient: 0 to 10% (2N NH₃in MeOH) in CH₂Cl₂) gave 4-

((((3R,4R)-4-((2-(benzyloxy)-5-(5-methoxypyridin-3-yl)-3-methyl-

1,7-naphthyridin-8-yl)amino)piperidin-3-yl)oxy)methyl)tetrahydro-

2H-thiopyran 1,1-dioxide. This derivative was dissolved in TFA (2

mL), and the resulting solution was reﬂuxed for 16 h, cooled to room

1

LCMS (high-pH method): t_R1.40 min, [M + H]⁺= 641.6. H NMR

(400 MHz, DMSO-d₆) δ ppm 9.30 (s, 1H), 9.23 (s, 1H), 8.91 (s, 2H),

7.89 (d, J = 1.0 Hz, 1H), 7.85 (s, 1H), 7.55 (d, J = 7.1 Hz, 2H), 7.38−

7.44 (m, 2H), 7.34 (s, 1H), 7.01−7.09 (m, 1H), 5.59−5.74 (m, 2H),

4.26−4.38 (m, 1H), 3.88−4.19 (m, 1H), 3.77−3.87 (m, 1H), 3.57−

3.67 (m, 2H), 3.47−3.55 (m, 1H), 3.38−3.45 (m, 1H), 3.20−3.27 (m,

1H), 3.07 (br s, 2H), 2.70−2.97 (m, 1H), 2.32 (s, 3H), 1.99−2.09 (m,

1H), 1.53−1.67 (m, 2H), 1.38−1.48 (m, 9H), 1.27−1.36 (m, 2H),

0.87−1.06 (m, 2H). Step 3: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-5-(pyrimidin-5-yl)-1,7-naphthyridin-8-yl)-

amino)-3-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxy-

late (120 mg, 0.187 mmol) in TFA (2 mL) was stirred at 80 °C for 2

h, cooled to room temperature, and concentrated in vacuo. Puriﬁcation

of the residue by MDAP (high-pH method) gave 3-methyl-5-

(pyrimidin-5-yl)-8-(((3R,4R)-3-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidin-4-yl)amino)-1,7-naphthyridin-2(1H)-one (41) (20

mg, 41%) as a colorless oil. LCMS (high-pH method): t_R0.57 min, [M

W

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

+ H]⁺= 451.3. H NMR (400 MHz, DMSO-d₆) δ ppm 9.23 (s, 1H),

(m, 2H), 1.52 (s, 9H), 0.84 (s, 9H), −0.09 (s, 6H). Step 5: a solution

of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methylquinolin-8-yl)-

amino)-3-((tert-butyldimethylsilyl)oxy)piperidine-1-carboxylate (1.60

g, 2.77 mmol) in THF (50 mL) at room temperature was treated

with TBAF (1 N in THF, 5.54 mL, 5.54 mmol), and the resulting

mixture was stirred at this temperature for 2 h and then concentrated

in vacuo. The residue was partitioned between EtOAc (100 mL) and

water (100 mL), and the layers were separated. The organic phase was

dried under MgSO₄and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (50 g column, gradient

0−50% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methylquinolin-8-yl)amino)-3-hydroxypiperidine-1-car-

boxylate (0.91g, 71%) as a colorless oil. LCMS (high-pH method): t_R

1.47 min, [M + H]⁺= 464. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.77

(d, J = 0.7 Hz, 1H), 7.50 (d, J = 7.3 Hz, 2H), 7.36−7.43 (m, 2H), 7.33

(d, J = 7.3 Hz, 1H), 7.19−7.25 (m, 1H), 7.01−7.07 (m, 1H), 6.84 (d, J

= 7.3 Hz, 1H), 5.50−5.59 (m, 2H), 5.42−5.49 (m, 1H), 4.28−4.39 (m,

1H), 3.92−4.22 (m, 1H), 3.58−3.69 (m, 1H), 3.41−3.53 (m, 1H),

2.83−3.02 (m, 2H), 2.59−2.73 (m, 1H), 2.41 (d, J = 1.0 Hz, 3H),

2.03−2.13 (m, 1H), 1.52 (s, 9H), 1.30−1.43 (m, 1H). Step 6: a

solution of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methylquinolin-8-

yl)amino)-3-hydroxypiperidine-1-carboxylate (420 mg, 0.906 mmol)

in THF (10 mL) at 0 °C was treated with potassium tert-butoxide

(224 mg, 1.99 mmol). The resulting mixture was stirred at this

temperature for 10 min, treated with (1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)methyl triﬂuoromethanesulfonate (537 mg, 1.81

mmol), and allowed to warm to room temperature over 2 h before

being diluted with water (10 mL). The aqueous phase was extracted

with EtOAc (10 mL). The organic layer was washed with water (10

mL), dried over MgSO₄, and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (25 g column, gradient: 0

to 100% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methylquinolin-8-yl)amino)-3-((1,1-dioxidotetrahydro-

2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (0.47 g, 85%) as

a pale green oil. LCMS (high-pH method): t_R1.51 min, [M + H]⁺=

610.5. Step 7: a solution of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-

methylquinolin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-

4-yl)methoxy)piperidine-1-carboxylate (0.47 g, 0.77 mmol) in CH₂Cl₂

(20 mL) at −10 °C was treated with NBS (0.123 g, 0.694 mmol), and

the resulting mixture was stirred at this temperature for 20 min before

being treated with a saturated sodium metabisulphite aqueous solution

(20 mL). The biphasic mixture was stirred at room temperature for 10

min, and the layers were separated. The organic phase was dried using

a phase separator and concentrated in vacuo. Puriﬁcation of the residue

by ﬂash chromatography on silica gel (25 g column, gradient: 0 to 50%

EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-

bromo-3-methylquinolin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)methoxy)piperidine-1-carboxylate (370 mg, 70%) as a

pale yellow gum. LCMS (high-pH method): t_R1.55 min, [M + H]⁺=

688/690. Step 8: a 20 mL microwave vial was charged with (5-

methylpyridin-3-yl)boronic acid (53.7 mg, 0.392 mmol), (3R,4R)-tert-

butyl 4-((2-(benzyloxy)-5-bromo-3-methylquinolin-8-yl)amino)-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate (180 mg, 0.261 mmol), K₂CO₃(144 mg, 1.04 mmol),

Pd(OAc)₂(5.9 mg, 0.026 mmol), and di((3S,5S,7S)-adamantan-1-

yl)(butyl)phosphine (cataCXium A) (9.4 mg, 0.026 mmol) and then

ﬁlled with 1,4-dioxane (4 mL) and water (2 mL). The resulting

mixture was degassed under vacuum over 20 min, quenched with

nitrogen, and stirred at 100 °C for 30 min under microwave irradiation

before being cooled to room temperature and concentrated in vacuo.

The residue was partitioned between CH₂Cl₂(20 mL) and water (20

mL), and the layers were separated. The organic phase was dried using

a hydrophobic frit and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (10 g column, gradient: 0

to 100% EtOAc in cyclohexane) gave (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-5-(5-methylpyridin-3-yl)quinolin-8-yl)amino)-

3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-

carboxylate (125 mg, 68%) as a pale yellow solid. LCMS (high-pH

method): t_R1.48 min, [M + H]⁺= 701.6. Step 9: a solution of

(3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-5-(5-methylpyridin-3-

8.86 (s, 2H), 7.76 (s, 1H), 7.59 (d, J = 1.2 Hz, 1H), 6.91−6.98 (m,

1H), 4.14−4.25 (m, 1H), 3.63−3.73 (m, 2H), 3.37−3.43 (m, 1H),

3.08−3.29 (m, 6H), 2.84−2.92 (m, 1H), 2.26−2.36 (m, 1H), 2.10 (d, J

= 1.0 Hz, 3H), 1.95−2.05 (m, 1H), 1.58−1.70 (m, 1H), 1.28−1.44 (m,

3H), 0.95−1.09 (m, 2H). Two NH not seen.

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-

3-yl)quinolin-2(1H)-one (42). Step 1: a solution of 2-methyl-3-

phenylacryloyl chloride (11 g, 60.9 mmol) in CH₂Cl₂(300 mL) at 0

°C under nitrogen was treated with NEt₃(9.34 mL, 67.0 mmol) and

then, after 10 min, with 2-bromoaniline (10.5 g, 60.9 mmol). The

resulting solution was stirred at this temperature for 4 h, washed

successively with water (300 mL), a 5% w/w Na₂CO₃aqueous

solution (300 mL), and a 1 N HCl aqueous solution (300 mL), dried

over MgSO₄, and concentrated in vacuo to give N-(2-bromophenyl)-2-

methyl-3-phenylacrylamide (18.5 g, 96%) as a dark brown oil that

crystallized on standing and that was used in the next step without

further puriﬁcation. Step 2: a solution of N-(2-bromophenyl)-2-

methyl-3-phenylacrylamide (16.0 g, 50.6 mmol) in chlorobenzene (40

mL) at 0 °C was treated with AlCl₃(40.5 g, 304 mmol), and the

resulting mixture was stirred at this temperature for 20 min and then at

120 °C for 2 h. The reaction mixture was cooled to room temperature

and then added very cautiously to ice (500 g). The mixture was

agitated thoroughly to ensure complete hydrolysis of aluminum

chloride. The mixture was then extracted with CH₂Cl₂(200 mL), and

the organic layer was dried over MgSO₄and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (330 g

column, 0 to 100% gradient EtOAc in cyclohexane) gave 8-bromo-3-

methylquinolin-2(1H)-one (6.9 g, 57%) as a pink solid. LCMS

(method formate): t_R0.82 min, [M + H]⁺= 238.0 (1 Br). H NMR

1

(400 MHz, CDCl₃) δ ppm 9.04 (br s, 1H), 7.66 (dd, J = 7.8, 1.1 Hz,

1H), 7.58 (d, J = 1.2 Hz, 1H), 7.47 (dd, J = 1.0, 7.8 Hz, 1H), 7.09 (t, J

= 7.8 Hz, 1H), 2.29 (d, J = 1.2 Hz, 3H). Step 3: a suspension of 8-

bromo-3-methylquinolin-2(1H)-one (1.0 g, 4.2 mmol) DMF (5 mL)

at room temperature was treated with K₂CO₃(0.71 g, 5.1 mmol), and

the resulting mixture was stirred at this temperature for 10 min before

(bromomethyl)benzene (0.55 mL, 4.6 mmol) was added. The

resulting mixture was then stirred for 72 h at room temperature.

The precipitate was ﬁltered oﬀ, the mother liquors were diluted with

water (50 mL) and EtOAc (50 mL), and the layers were separated.

The organic phase was washed with brine (3 × 50 mL), dried over

Na₂SO₄, and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, 0 to 100% gradient EtOAc

in cyclohexane) gave 2-(benzyloxy)-8-bromo-3-methylquinoline (1.17

g, 85%) as a pink solid and 1-benzyl-8-bromo-3-methylquinolin-

2(1H)-one (40 mg, 3%) as a milky white gum. LCMS (method

formate): t_R1.57 min, [M + H]⁺= 328.0 (1 Br).¹H NMR (400 MHz,

CDCl₃) δ ppm 7.72 (dd, J = 7.8, 1.7 Hz, 1H), 7.55 (d, J = 1.2 Hz, 1H),

7.47 (dd, J = 7.7, 1.5 Hz, 1H), 7.18−7.31 (m, 3H), 7.10 (m, 2H), 7.04

(dd, J = 7.7, 7.7 Hz, 1H), 6.06 (s, 2H), 2.29 (d, J = 1.2 Hz, 3H). Step

4: a mixture of (3R,4R)-tert-butyl 4-amino-3-((tert-butyldimethylsilyl)-

oxy)piperidine-1-carboxylate (1.21 g, 3.66 mmol), 2-(benzyloxy)-8-

bromo-3-methylquinoline (1.0 g, 3.0 mmol), sodium tert-butoxide

(0.878 g, 9.14 mmol), BrettPhos (0.164 g, 0.305 mmol), and

BrettPhos precatalyst (0.243 g, 0.305 mmol) in THF (2 mL) was

stirred at room temerature for 1 h, stirred at 60 °C for 3 h, cooled to

room temperature, and diluted with a saturated NH₄Cl aqueous

solution. The aqueous phase was extracted with EtOAc (2 × 50 mLl),

and the combined organics were dried over MgSO₄and concentrated

in vacuo. Puriﬁcation of the residue by ﬂash chromatography on silica

gel (50 g column, gradient: 0−25% EtOAc in cyclohexane) gave

(3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methylquinolin-8-yl)amino)-3-

((tert-butyldimethylsilyl)oxy)piperidine-1-carboxylate (1.70 g, 97%) as

a pale yellow foam. LCMS (high-pH method): t_R1.90 min, [M + H]⁺

= 578. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.76 (d, J = 0.7 Hz, 1H),

7.49 (s, 2H), 7.39 (s, 2H), 7.30−7.35 (m, 1H), 7.17−7.23 (m, 1H),

6.93−6.99 (m, 1H), 6.72−6.77 (m, 1H), 5.62−5.68 (m, 1H), 5.55 (d, J

= 8.8 Hz, 2H), 3.72−3.93 (m, 2H), 3.59−3.67 (m, 1H), 3.48−3.57 (m,

1H), 3.16−3.27 (m, 1H), 3.05−3.14 (m, 1H), 2.40 (s, 3H), 1.60−1.76

X

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Article

yl)quinolin-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-

yl)methoxy)piperidine-1-carboxylate (125 mg, 0.178 mmol) in TFA (1

mL) was reﬂuxed for 2 h, cooled to room temperature, and

concentrated in vacuo. The residue was dissolved in MeOH and

loaded onto a 5 g SCX-2 cartridge, which was washed with MeOH (20

mL) and eluted with a 2 N NH₃solution in MeOH. The ammoniac

fractions were concentrated in vacuo to give 8-(((3R,4R)-3-((1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidin-4-yl)amino)-

3-methyl-5-(5-methylpyridin-3-yl)quinolin-2(1H)-one (42) (90 mg,

99%) as a yellow solid. LCMS (high-pH method): t_R0.68 min, [M +

H]⁺= 511, ¹H NMR (400 MHz, CDCl₃) δ ppm 11.70 (br s, 1H), 8.51

(d, J = 1.5 Hz, 1H), 8.46 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 1.2 Hz, 1H),

7.52 (s, 1H), 6.99−7.06 (m, 2H), 4.24 (br s, 1H), 3.58−3.65 (m, 1H),

3.36−3.56 (m, 4H), 3.20 (br s, 1H), 2.94−3.14 (m, 5H), 2.57−2.66

(m, 1H), 2.55−2.57 (m, 1H), 2.52 (dd, J = 11.5, 9.5 Hz, 1H), 2.46 (s,

3H), 2.07−2.31 (m, 4H), 1.75−1.90 (m, 3H), 1.53−1.62 (m, 2H).

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-

3-yl)quinolin-2(1H)-one (43). This compound was obtained in an

anlogous manner as that for compound 42 following the separation of

the two enantiomers of (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-

methylquinolin-8-yl)amino)-3-hydroxypiperidine-1-carboxylate (step

5, compound 42), which was performed as follows: The racemic

mixture was separated by chiral HLPC. The HPLC analysis was carried

out on a Chiralpak IC (4.6 mm i.d. × 25 cm), using 25% EtOH in

heptane at a ﬂow rate of 1 mL/min. The material (910 mg) was

dissolved in a mixture of EtOH/heptane (2:1) (1 mL). The

puriﬁcation was carried out on a Chiralpak IC (30 mm × 25 cm),

using 25% EtOH in heptane at a ﬂow rate of 30 mL/min, and 1 mL of

solution was injected at a time. The appropriate fractions were

combined and concentrated in vacuo to give the fastest running

enantiomer, (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methylquinolin-8-

yl)amino)-3-hydroxypiperidine-1-carboxylate (422 mg), and the

slowest running enantiomer, (3S,4S)-tert-butyl 4-((2-(benzyloxy)-3-

methylquinolin-8-yl)amino)-3-hydroxypiperidine-1-carboxylate (423

mg).

8-(((3R,4R)-3-(((2R,4R,6S)-2,6-Dimethyl-1,1-dioxidotetrahy-

dro-2H-thiopyran-4-yl)methoxy)piperidin-4-yl)amino)-3-meth-

yl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one (44).

Step 1: a solution of 4-(hydroxymethyl)tetrahydro-2H-thiopyran 1,1-

dioxide (1.0 g, 6.1 mmol) in CH₂Cl₂(30 mL) at room temperature

was treated with imidazole (0.497 g, 7.31 mmol) and DMAP (0.074 g,

0.61 mmol). The mixture was stirred at this temperature for 20 min;

then, TBDMSCl (1.10 g, 7.31 mmol) was added, and the resulting

suspension was stirred for 2 h. The organic phase was then washed

with water (30 mL), dried using a phase separator, and concentrated in

vacuo. Puriﬁcation of the residue by ﬂash chromatography on silica gel

(25 g column, gradient: 0 to 50% EtOAc in cyclohexane) gave 4-

(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-thiopyran 1,1-di-

oxide (1.62 g, 96%) as a colorless oil. Step 2: a solution of 4-(((tert-

butyldimethylsilyl)oxy)methyl)tetrahydro-2H-thiopyran 1,1-dioxide

(1.4 g, 5.0 mmol) in THF (10 mL) at −78 °C under nitrogen was

treated with LiHMDS (1 N in THF, 6.54 mL, 6.54 mmol). The

resulting mixture was stirred at this temperature for 30 min before

being treated with MeI (0.503 mL, 8.04 mmol). The mixture was

stirred at −78 °C for 2 h and was then allowed to warm to room

temperature. The solution was diluted with water (30 mL), and the

aqueous phase was extracted with EtOAc (20 mL). The organic phase

was dried over MgSO₄and concentrated in vacuo to give (2R,6S)-4-

(((tert-butyldimethylsilyl)oxy)methyl)-2,6-dimethyltetrahydro-2H-thi-

opyran 1,1-dioxide (contaminated with a mixture of monomethylated

adducts) (1.45 g, 95%) as a pale yellow oil, which was used in the next

step without further puriﬁcation. Step 3: a solution of (2R,6S)-4-

(((tert-butyldimethylsilyl)oxy)methyl)-2,6-dimethyltetrahydro-2H-thi-

opyran 1,1-dioxide (0.50 g, 1.7 mmol) in THF (10 mL) at room

temperature was treated with a 2 N HCl aqueous solution (10 mL),

and the resulting mixture was stirred at this temperature for 18 h and

then concentrated in vacuo to give (2R,6S)-4-(hydroxymethyl)-2,6-

dimethyltetrahydro-2H-thiopyran 1,1-dioxide (0.186 g, 61%) as a

colorless solid (contaminated with a mixture of monomethylated

adducts), which was used in the next step without further puriﬁcation.

Step 4: a solution of (2R,6S)-4-(hydroxymethyl)-2,6-dimethyltetrahy-

dro-2H-thiopyran 1,1-dioxide (0.186 g, 1.04 mmol) in CH₂Cl₂(10

mL) under nitrogen was cooled using an acetone/ice bath (−10 °C)

then was treated with pyridine (0.093 mL, 1.15 mmol) and Tf₂O

(0.194 mL, 1.15 mmol) dropwise. The resulting mixture was stirred at

this temperature for 1 h and then allowed to warm to room

temperature. The organic phase was washed with brine (10 mL), dried

using a phase separator, and concentrated in vacuo to give ((2R,6S)-

2,6-dimethyl-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methyl triﬂuor-

omethanesulfonate (0.32 g, 99%) as a pale yellow oil (contaminated

with a mixture of monomethylated adducts), which was used in the

next step without further puriﬁcation. Step 5: a solution of (3R,4R)-

tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-

hydroxypiperidine-1-carboxylate (0.30 g, 0.65 mmol) in THF (10 mL)

at 0 °C under nitrogen was treated with tBuOK (0.152 g, 1.36 mmol),

and the resulting mixture was stirred at this temperature for 10 min

before being treated with a solution of ((2R,6S)-2,6-dimethyl-1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)methyl triﬂuoromethanesulfo-

nate (0.301 g, 0.969 mmol) in THF (10 mL). The resulting mixture

was stirred at 0 °C for 1 h and then at room temperature for 30 min

before being quenched with water. The aqueous phase was extracted

with EtOAc. The organic phase was washed with water, dried over

MgSO₄, and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (25 g column, gradient 0 to 70% EtOAc

in hexanes) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-

naphthyridin-8-yl)amino)-3-(((2R,6S)-2,6-dimethyl-1,1-dioxidotetra-

hydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (0.19 g,

47%) as a pale yellow gum contaminated with a mixture of

monomethylated adducts. LCMS (high-pH method): t_R1.43 min,

[M + H]⁺= 639.5. Step 6: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-(((2R,6S)-2,6-

dimethyl-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidine-1-carboxylate (0.19 g, 0.30 mmol) in CH₂Cl₂(10 mL) at

−10 °C was treated with NBS (0.049 g, 0.27 mmol), and the resulting

mixture was stirred at this temperature for 40 min and then treated

with a saturated sodium metabisulphite aqueous solution. The biphasic

mixture was allowed to warm to room temperature and stirred for 10

min. The layers were separated, and the organic phase was dried using

a phase separator and concentrated in vacuo to give (3R,4R)-tert-butyl

4-((2-(benzyloxy)-5-bromo-3-methyl-1,7-naphthyridin-8-yl)amino)-3-

(((2R,6S)-2,6-dimethyl-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidine-1-carboxylate (180 mg, 84%) as a pale yellow gum

(contaminated with a mixture of monomethylated adducts), which was

used in the next step without further puriﬁcation. LCMS (high-pH

method): t_R1.57 min, [M + H]⁺= 719.4 (1 Br). Step 7: a 20 mL

microwave vial was charged with (5-methylpyridin-3-yl)boronic acid

(0.070 g, 0.51 mmol), (3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-

3-methyl-1,7-naphthyridin-8-yl)amino)-3-((2-methyl-1,1-dioxidotetra-

hydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (0.18 g,

0.26 mmol), K₂CO₃(0.141 g, 1.02 mmol), Pd(OAc)₂(5.74 mg,

0.026 mmol), and di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine

(cataCXium A) (9.2 mg, 0.026 mmol) and then ﬁlled with 1,4-

dioxane (4 mL) and water (2 mL). The resulting mixture was degassed

under vacuum for 20 min and quenched with nitrogen before being

stirred at 100 °C for 30 min under microwave irradiation.. The mixture

was then cooled to room temperature and concentrated in vacuo.

Puriﬁcation of the residue by MDAP (high-pH method) gave (3R,4R)-

tert-butyl 4-((2-(benzyloxy)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-

naphthyridin-8-yl)amino)-3-(((2R,6S)-2,6-dimethyl-1,1-dioxidotetra-

hydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (140 mg,

76%) contaminated with a mixture of monomethylated adducts. The

mixture was further puriﬁed by chiral HLPC. The HPLC analysis was

carried out on a Chiralpak IB (4.6 mm i.d. × 25 cm), using 10% EtOH

(+0.2% isopropylamine) in heptane at a ﬂow rate of 1 mL/min. The

material (140 mg) was dissolved in a mixture of EtOH (100 mg in

mL). The puriﬁcation was carried out on a Chiralpak IB (2 cm × 25

cm), using 10% EtOH (+0.2% isopropylamine) in heptane at a ﬂow

rate of 20 mL/min, and 1 mL of solution was injected. The

appropriate fractions were combined and concentrated under reduced

Y

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Article

pressure to give (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-5-(5-

methylpyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-3-(((2R,4R,6S)-2,6-

dimethyl-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidine-1-carboxylate (80 mg, 43%). LCMS (high-pH method): t_R

1.43 min, [M + H]⁺= 730.6. Step 8: a solution of (3R,4R)-tert-butyl 4-

((2-(benzyloxy)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-

8-yl)amino)-3-(((2R,4r,6S)-2,6-dimethyl-1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)methoxy)piperidine-1-carboxylate (70 mg, 0.096

mmol) in TFA (2 mL) was reﬂuxed for 2 h and then cooled to

room temperature and concentrated in vacuo. Puriﬁcation of the

residue by MDAP (high-pH method) gave 8-(((3R,4R)-3-

(((2R,4r,6S)-2,6-dimethyl-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-3-yl)-

1,7-naphthyridin-2(1H)-one (44) (20 mg, 39%) as a bright yellow

solid. LCMS (method TFA): t_R0.47 min, [M + H]⁺= 540. ¹H NMR

(400 MHz, CDCl₃) δ ppm 13.2 (br s, 1H), 8.53 (d, J = 1.5 Hz, 1H),

8.48 (d, J = 1.7 Hz, 1H), 7.90−7.94 (m, 1H), 7.75 (d, J = 1.0 Hz, 1H),

7.54 (s, 1H), 6.75 (d, J = 6.4 Hz, 1H), 4.52 (br s, 1H), 3.46−3.59 (m,

2H), 3.32−3.45 (m, 2H), 3.09−3.17 (m, 1H), 2.77−2.92 (m, 3H),

2.63−2.71 (m, 1H), 2.47 (s, 3H), 2.34 (d, J = 0.7 Hz, 3H), 2.19−2.27

(m, 1H), 1.84−1.94 (m, 3H), 1.60−1.76 (m, 2H), 1.30−1.54 (m, 2H),

1.23 (d, J = 6.8 Hz, 3H), 1.15 (d, J = 6.8 Hz, 3H).

an NaCl ice bath and then treated with NBS (10.6 mg, 0.060 mmol),

and the resulting mixture was stirred at this temperature for 30 min.

NBS (3.6 mg, 0.020 mmol) was further added, and the mixture was

stirred at the same temperature for 30 min before being treated with a

saturated sodium metabisulﬁte aqueous solution (5 mL). The reaction

mixture was stirred for 20 min and then warmed to room temperature.

The layers were separated, and the organic phase was dried using a

hydrophobic frit and concentrated in vacuo to give (3R,4R)-tert-butyl

4-((2-(benzyloxy)-5-bromo-3-ethyl-1,7-naphthyridin-8-yl)amino)-3-

((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-car-

boxylate (59.4 mg, 88%) as a yellow oil, which was used in the next

step without further puriﬁcation. LCMS (method formic): t_R1.46 min,

[M + H]⁺= 705.6 (1 Br). Step 6: a 5 mL microwave vial was charged

with (5-methylpyridin-3-yl)boronic acid (15 mg, 0.11 mmol),

(3R,4R)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-ethyl-1,7-naphthyri-

din-8-yl)amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidine-1-carboxylate (59 mg, 0.084 mmol), Pd(OAc)₂

(2.0 mg, 8.9 μmol), K₂CO₃(51 mg, 0.37 mmol), and di((3S,5S,7S)-

adamantan-1-yl)(butyl)phosphine (cataCXium A) (3.0 mg, 8.4 μmol)

and then ﬁlled with 1,4-dioxane (2 mL) and water (1 mL), and the

resulting mixture was degassed by bubbling nitrogen through it for 30

min. The mixture was then stirred under microwave irradiation at 100

°C for 30 min before being cooled to room temperature and

concentrated in vacuo. The residue was partitioned between CH₂Cl₂(8

mL) and water (8 mL), and the layers were separated. The organic

phase was dried using a hydrophobic frit and then concentrated in

vacuo. Puriﬁcation of the residue by ﬂash chromatography on silica gel

(25 g column, gradient: 12 to 63% (3:1 EtOAc/EtOH) in

cyclohexane) gave (3R,4R)-tert-butyl 4-((2-(benzyloxy)-3-ethyl-5-(5-

methylpyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (25.6

mg, 42%) as a yellow oil. LCMS (high-pH method): t_R1.45 min,

[M + H]⁺= 717.7. Step 7: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-ethyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-8-yl)-

amino)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidine-1-carboxylate (25 mg, 0.036 mmol) in TFA (1 mL) was

reﬂuxed at 80 °C for 2 h, cooled to room temperature, and

concentrated in vacuo. The residue was again dissolved in TFA (1

mL), and the mixture was reﬂuxed for another 3 h before being cooled

to room temperature and concentrated in vacuo. The residue was

loaded on to a 500 mg SCX-2 cartridge, which was washed with

MeOH (5 mL) and then eluted with a 2 N NH₃solution in MeOH.

The ammoniac fractions were combined and concentrated in vacuo to

give 8-(((3R,4R)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-ethyl-5-(5-methylpyridin-3-yl)-1,7-

naphthyridin-2(1H)-one (45) (12.1 mg, 64%) as a yellow solid. LCMS

(method formic): t_R0.51 min, [M + H]⁺= 526. ¹H NMR (400 MHz,

CD₃OD) δ ppm 8.46 (d, J = 1.5 Hz, 1H), 8.41 (d, J = 1.7 Hz, 1H),

7.75−7.79 (m, 2H), 7.61 (s, 1H), 4.30−4.38 (m, 1H), 3.60 (dd, J =

9.2, 5.5 Hz, 1H), 3.38−3.46 (m, 2H), 3.05−3.12 (m, 1H), 2.82−3.01

(m, 4H), 2.72−2.81 (m, 1H), 2.64 (q, J = 7.8 Hz, 2H), 2.54−2.60 (m,

1H), 2.47 (s, 4H), 2.16−2.25 (m, 1H), 1.93−2.02 (m, 2H), 1.75 (ddd,

J = 8.4, 5.6, 2.9 Hz, 1H), 1.58−1.70 (m, 3H), 1.19 (t, J = 7.5 Hz, 3H).

Three NH not seen.

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-ethyl-5-(5-methylpyridin-3-

yl)-1,7-naphthyridin-2(1H)-one (45). Step 1: (E)-ethyl 2-((3-((tert-

butoxycarbonyl)amino)-2-chloropyridin-4-yl)methylene)butanoate

was obtained from tert-butyl (2-chloro-4-formylpyridin-3-yl)carbamate

in an anaogous manner as that for (E)-ethyl 3-(3-((tert-

butoxycarbonyl)amino)-2-chloropyridin-4-yl)-2-methyl acrylate (see

synthesis of 7a in preceding article⁶) using ethyl 2-

1

(diethoxyphosphoryl)butanoate (15 g, 57%). H NMR (400 MHz,

DMSO-d₆) δ ppm 8.33 (d, J = 4.8 Hz, 1H), 7.39 (s, 1H), 7.35 (d, J =

4.8 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.27 (d, J = 7.5 Hz, 2H), 1.39

(br s, 7H), 1.25 (t, J = 7.1 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H). Step 2:

(E)-ethyl 2-((3-amino-2-chloropyridin-4-yl)methylene)butanoate was

obtained from (E)-ethyl 2-((3-((tert-butoxycarbonyl)amino)-2-chlor-

opyridin-4-yl)methylene)butanoate using the same procedure as that

for the synthesis of (E)-ethyl 3-(3-amino-2-chloropyridin-4-yl)-2-

methyl acrylate from ethyl 3-(3-((tert-butoxycarbonyl)amino)-2-

chloropyridin-4-yl)-2-methyl acrylate (see synthesis of 7a in preceding

article⁶) (8.1 g, 75%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.63 (d,

J = 1.0 Hz, 1H), 7.34 (s, 1H), 6.98 (d, J = 1.0 Hz, 1H), 5.50 (s, 2H),

4.19−4.25 (m, 2H), 2.26−2.34 (m, 2H), 1.29 (t, J = 1.0 Hz, 3H), 1.03

(t, J = 1.0 Hz, 3H). Step 3: 8-chloro-3-ethyl-1,7-naphthyridin-2(1H)-

one was obtained from (E)-ethyl 2-((3-amino-2-chloropyridin-4-

yl)methylene)butanoate using the same procedure as that for the

synthesis of 8-chloro-3-methyl-1,7-naphthyridin-2(1H)-one (7a) from

(E)-ethyl 3-(3-amino-2-chloropyridin-4-yl)-2-methyl acrylate (5 g,

1

87%). H NMR (400 MHz, DMSO-d₆) δ ppm 11.35 (s, 1H), 8.13

(d, J = 5.0 Hz, 1H), 7.83 (s, 1H), 7.64 (d, J = 5.0 Hz, 1H), 2.53−2.60

(m, 2H), 1.19 (t, J = 7.5 Hz, 3H). Step 4: a solution of (3R,4R)-tert-

butyl 4-amino-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidine-1-carboxylate (249 mg, 0.686 mmol) in THF (4 mL) was

treated with 2-(benzyloxy)-8-chloro-3-ethyl-1,7-naphthyridine (248

mg, 0.830 mmol), sodium tert-butoxide (203 mg, 2.11 mmol),

Pd₂(dba)₃(34 mg, 0.037 mmol), and BrettPhos (39 mg, 0.074 mmol).

The resulting mixture was stirred at room temperature for 1 h and was

then reﬂuxed for 3 h before being cooled to room temperature and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (50 g column, gradient: 15 to 70%

EtOAc in cyclohexane) gave a ﬁrst residue, which was further puriﬁed

by ﬂash chromatography on silica gel (50 g column, gradient: 15 to

70% EtOAc in cyclohexane) to give (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-ethyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (69.1

mg, 16%) as a yellow oil. LCMS (method formic): t_R1.04 min, [M

+ H]⁺= 625.6. Step 5: a solution of (3R,4R)-tert-butyl 4-((2-

(benzyloxy)-3-ethyl-1,7-naphthyridin-8-yl)amino)-3-((1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)methoxy)piperidine-1-carboxylate (6.1

mL, 0.096 mmol) in CH₂Cl₂(5 mL) was cooled to −10 °C using

8-(((3R,4R)-3-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-

methoxy)-1-methylpiperidin-4-yl)amino)-3-methyl-5-(5-meth-

ylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one (46). A solution of 8-

(((3R,4R)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-

piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naph-

thyridin-2(1H)-one (110 mg, 0.215 mmol) in CHCl₃was treated with

formic acid (11 μL, 0.28 mmol) and with a formaldehyde solution in

water, containing 10−15% MeOH as stabilizer (37% w/w, 19 μL, 0.26

mmol). The resulting mixture was stirred under microwave irradiation

at 80 °C for 2 h, cooled to room temperature, and concentrated in

vacuo. Puriﬁcation of the residue by MDAP (high-pH method) gave 8-

(((3R,4R)-3-((1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy)-1-

methylpiperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-

naphthyridin-2(1H)-one (46) (67 mg, 59%) as a yellow solid. LCMS

1

(method TFA): t_R0.77 min, [M + H]⁺= 526. H NMR (400 MHz,

CDCl₃) δ ppm 13.2 (br s, 1H), 8.52 (d, J = 1.5 Hz, 1H), 8.46 (d, J =

Z

DOI: 10.1021/acs.jmedchem.5b00773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.7 Hz, 1H), 7.90 (s, 1H), 7.73 (d, J = 1.2 Hz, 1H), 7.54 (d, J = 0.7 Hz,

1H), 6.64 (d, J = 7.6 Hz, 1H), 4.32−4.43 (m, 1H), 3.70 (td, J = 9.1, 4.3

Hz, 1H), 3.55−3.61 (m, 1H), 3.48−3.54 (m, 1H), 3.17 (dd, J = 10.9,

3.3 Hz, 1H), 2.75−2.98 (m, 6H), 2.47 (s, 3H), 2.38 (s, 3H), 2.34 (d, J

= 1.0 Hz, 3H), 2.21−2.32 (m, 3H), 2.16 (t, J = 10.1 Hz, 1H), 1.98−

2.11 (m, 2H), 1.66−1.86 (m, 2H).

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ASSOCIATED CONTENT

* Supporting Information

■

(6) Demont, E. H.; Chung, C.; Furze, R.; Grandi, P.; Michon, A. M.;

Wellaway, C.; Barrett, N.; Bridges, A. M.; Craggs, P. D.; Diallo, H.;

Dixon, D. P.; Douault, C.; Emmons, A.; Jones, E. J.; Karamshi, B.;

Locke, K.; Mitchell, D. J.; Mouzon, B. H.; Prinjha, R. K.; Roberts, A.

D.; Sheppard, R. J.; Watson, R. J.; Bamborough, P. Fragment-based

discovery of low-micromolar ATAD2 bromodomain inhibitors. J. Med.

Chem. 2015, 58, 5649.

S

Table S1, parts 1 and 2: Full data for exempliﬁed compounds in

main tables. Table S2: Selectivity proﬁle of compound 46 in the

BromoSCAN panel. Table S3: Data collection and reﬁnement

statistics for ATAD2 X-ray structures. Table S4: Data collection

and reﬁnement statistics for BRD4 BD1 structure. Figure S1:

Correlation plots between ATAD2 assays. Figure S2: KAc site

amino acids of ATAD2, ATAD2B, BRD4 BD1, and BRD4 BD2

bromodomains. Figure S3: OMIT maps for ATAD2 and BRD4

BD1 X-ray structures. Figure S4: GSK3190320, the synthetic

ligand used in the ATAD2 TR-FRET assay. The Supporting

Information is available free of charge on the ACS Publications

website at DOI: 10.1021/acs.jmedchem.5b00773.

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AUTHOR INFORMATION

Corresponding Authors

*(P.B.) E-mail: Paul.A.Bamborough@gsk.com. Phone: +44

1438 763246. Fax: +44 1438 763352.

■

*(E.H.D.; for materials or collaboration) E-mail: Emmanuel.H.

Demont@gsk.com. Phone: +44 1438 764319. Fax +44 1438

768302.

Present Address

^⊥(R.J.S.) Oncology Innovative Medicines and Early Develop-

ment, AstraZeneca, Cambridge Science Park, Milton Road,

Cambridge CB4 OWG, U.K.

Notes

The authors declare the following competing ﬁnancial

interest(s): All authors are current or previous employees of

GSK.

ACKNOWLEDGMENTS

Thanks are extended to Tony Cooper (array design and

chemistry) and Antonia Lewis (PBMC assay).

■

(12) Poncet-Montange, G.; Zhan, Y.; Bardenhagen, J. P.; Petrocchi,

A.; Leo, E.; Shi, X.; Lee, G. R.; Leonard, P. G.; Geck Do, M. D.;

Cardozo, M. K.; Andersen, M. G.; Palmer, J. N.; Jones, W. S.; Ladbury,

P. J. E. Observed bromodomain flexibility reveals histone peptide- and

small molecule ligand-compatible forms of ATAD2A. Biochem. J. 2015,

466, 337−346.

(13) Li, X.; Russell, R. K.; Spink, J.; Ballentine, S.; Teleha, C.;

Branum, S.; Wells, K.; Beauchamp, D.; Patch, R.; Huang, H.; Player,

M.; Murray, W. Process Development for Scale-Up of a Novel 3, 5-

Substituted Thiazolidine-2, 4-dione Compound as a Potent Inhibitor

for Estrogen-Related Receptor 1. Org. Process Res. Dev. 2014, 18, 321−

330.

ABBREVIATIONS USED

■

ANCCA, AAA nuclear coregulator cancer-associated protein;

ATAD2, ATPase family AAA domain containing 2; BET,

bromodomain and extra terminal domain; BRD4, bromodo-

main-containing protein 4; BRPF1/2/3, bromodomain and

PHD ﬁnger containing 1/2/3; CECR2, cat eye syndrome

chromosome region, candidate 2; KAc, acetyl lysine. pIC₅₀,

−log₁₀IC₅₀; SPR, surface plasmon resonance; TAF, TATA box

binding protein (TBP)-associated factor

(14) Fink, B. E., Gavai, A. V., Vite, G. D., Chen, P., Mastalerz, H.,

Norris-Drouin, J., Tokarski, J. S., Zhao, Y., Han, W. C. Pyrrolotriazine

compounds as kinase inhibitors. PCT Int. Appl WO2005066176A1,

2005.

(15) The BROMOscan panel, courtesy of DiscoveRx Corp. www.

discoverx.com.

(16) Further details of the application of Cellzome bead-based

chemoproteomics technology to bromodomain-containing proteins

will be the subject of future publications.

(17) Bergamini, G.; Bell, K.; Shimamura, S.; Werner, T.; Cansfield,

A.; Muller, K.; Perrin, J.; Rau, C.; Ellard, K.; Hopf, C.; Doce, C.;

Leggate, D.; Mangano, R.; Mathieson, T.; O’Mahony, A.; Plavec, I.;

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