Aiming to Miss a Moving Target: Bromo and Extra Terminal Domain (BET) Selectivity in Constrained ATAD2 Inhibitors

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Aiming to Miss a Moving Target: Bromo and Extra Terminal Domain

(BET) Selectivity in Constrained ATAD2 Inhibitors

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

Robert J. Watson,^‡Darren J. Mitchell,^‡Heather Barnett,^§Rab K. Prinjha,^‡Christina Rau,^∥

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

‡

§

^†Molecular Discovery Research, Epigenetics Discovery Performance Unit, and Flexible Discovery Unit, GlaxoSmithKline

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

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

S

* Supporting Information

ABSTRACT: ATAD2 is a cancer-associated protein whose

bromodomain has been described as among the least

druggable of its class. In our recent disclosure of the ﬁrst

chemical probe against this bromodomain, GSK8814 (6), we

described the use of a conformationally constrained methoxy

piperidine to gain selectivity over the BET bromodomains.

Here we describe an orthogonal conformational restriction

strategy of the piperidine ring to give potent and selective

tropane inhibitors and show structural insights into why this

was more challenging than expected. Greater understanding of

why diﬀerent rational approaches succeeded or failed should

help in the future design of selectivity in the bromodomain

family.

far, drug discovery eﬀorts for oncology have focused on the

ATAD2 bromodomain.^24,25The discovery that molecules

identiﬁed through phenotypic screens act by inhibition of the

bromodomains of the BET family demonstrated the

ligandability of bromodomains and highlighted their potential

in a wide range of clinical indications, including oncology and

inﬂammation.²⁶⁻²⁸A number of drug-like BET bromodomain

inhibitors have now been reported, and over 20 clinical trials

are underway studying their eﬀects in oncology and

inﬂammatory diseases.²⁹A growing number of tool molecules

have been reported to inhibit the bromodomains of non-BET

proteins, including but not limited to CBP/EP300, BRD7/9,

BRPF1, and SMARCA2/4.³⁰⁻³²Because of the potent

pharmacology of even quite weak BET inhibitors,³³cellular

chemical probes of other bromodomains require exceptional

selectivity. We have typically tried to reach 100−1000-fold

selectivity. However, because of the conserved nature of

bromodomain KAc sites, this is not always easily achieved.

Greater understanding of which rational attempts to engineer

greater selectivity succeed, and why they can unexpectedly fail,

provides insights to inform future eﬀorts.

INTRODUCTION

■

ATAD2, also known as ATAD2A or ANCCA (AAA nuclear

coregulator cancer-associated protein), is a nuclear protein

normally expressed in germ cells and embryonic stem cells with

a key role in chromatin remodelling.¹Since its identiﬁcation as

a target gene in breast cancer,^2,3ATAD2 has been shown to be

highly expressed in a wide variety of unrelated cancers with

increased expression levels correlating with disease recurrence

and poor clinical prognosis.⁴Potential oncology indications

include lung adenocarcinoma,⁵hepatocellular carcinoma,^6,7

colorectal,⁸ovarian,⁹and endometrial cancer¹⁰as well as

osteosarcoma¹¹and gastric cancer.¹²

As an oncology target, ATAD2 is supported by down-

regulation of protein expression using siRNA technology. This

has implicated the role of ATAD2 in pathways including

apoptosis, cell survival, proliferation, and migration.^2,4,13−16

Mechanistically, ATAD2 has been shown to function as a

coactivator for a variety of transcription factors including

MYC, estrogen/androgen receptors, and E2F family members,

directing transcription of a number of genes involved in cell

proliferation and survival, including cyclins, Cdk2, and

kinesins.¹⁷⁻²¹Furthermore, it has been demonstrated that

ATAD2 is recruited to newly synthesized histones during DNA

replication via a direct interaction with diacetylation

modiﬁcations at K5 and K12 on histone H4.^22,23

We^34,35and others³⁶⁻³⁸have identiﬁed small molecule

inhibitors of the ATAD2/ATAD2B bromodomain to probe its

functional role in disease and to assess if its inhibition was

ATAD2 possesses a bromodomain and an ATPase

Received: May 30, 2018

associated with diverse cellular activities (AAA+) domain. So

A

DOI: 10.1021/acs.jmedchem.8b00862

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Journal of Medicinal Chemistry

Article

Table 1. Structure and Proﬁle of Our Reported ATAD2 Inhibitors

compd

R

X

ATAD2 pIC₅₀

BRD4 BD1 pIC₅₀

selectivity (fold)

1

2

3

4

5

6

5.6

6.9

6.7

6.9

7.3

5.4

6.2

5.8

4.8

3.8

4.6

2

5

8

125

1300

500

H

OMe

CH₂

SO₂

CF₂

Figure 1. Compound 6 bound to ATAD2 and BRD4 (same orientation) illustrating the conformational basis for selectivity. In both proteins, the

residues of the RVF/WPF motif are shown in pale yellow and the gatekeeper residue in salmon. In ATAD2 (A, PDB 5LJ0), the piperidine ring is

triequatorial, positioning the C3′-substituent upon the RVF shelf where its CF₂group interacts with R1077. In BRD4 (B, PDB 5LJ1), the triaxial

piperidine geometry moves the C3′ substituent away from the WPF shelf into solvent.

suﬃcient to reproduce the promising results seen with siRNA

knockdown. Starting from a fragment hit, we identiﬁed potent,

cell permeable, highly selective ATAD2 inhibitors such as

compound 6 (Table 1).³⁹This uses a methoxy C5′-substituent

to constrain the piperidine ring to an equatorial geometry in

order to gain selectivity against the BET bromodomains. In

this article, we present results from an alternative means of

conformational restriction using 2-carbon-bridged piperidine

(tropane) derivatives. This approach led to the identiﬁcation of

compounds with good potency and selectivity over the BET

bromodomains. It also provided additional rationale for why

compounds such as 6 have exceptional selectivity against BET.

As previously reported,^34,35we used structure-based design

to increase the potency and selectivity for ATAD2 over BRD4

BD1 (a representative of the eight bromodomains of this

family, see Supporting Information, Tables S2 and S3) starting

from compound 1 to give inhibitors such as 2 and 3 (Table 1).

In particular we exploited the diﬀerent interactions between

the C3′-substituent and the RVF shelf in ATAD2 and the WPF

shelf in BET. We reasoned that certain polar analogues of this

cyclohexyl ring would interact better with the arginine side

chains Arg1007 and Arg1077 found in ATAD2 than with the

analogous tryptophan and methionine of the BET family

(Trp81 and Met149 in BRD4 BD1), hence providing

selectivity. This was indeed found to be the case (compare

compounds such as 4 to 3).³⁵Crystal structures of the

analogous quinolinone series in ATAD2 and BET revealed

unexpected features of their binding modes.³⁹In ATAD2, the

piperidine lies in a trans-diequatorial conformation, making

speciﬁc interactions with Arg1077. Unexpectedly, in BRD4

BD1 the piperidine ring adopts a trans-diaxial conformation to

remove the polar sulfone C3′-group from the WPF shelf.

Selectivity for ATAD2 over BRD4 BD1 was increased further

by stabilizing the equatorial conformation versus the axial. This

was achieved by the introduction of a C5′-methoxy substituent

cis to the C3′ ether, giving 5. This group creates a 1,3 steric

strain in the putative triaxial conformation, disfavoring binding

to BET, with limited impact on the triequatorial conformation

seen in ATAD2. A ﬁnal modiﬁcation (isosteric substitution of

the SO₂group with CF₂) dramatically increased the passive

permeability of our inhibitors, leading to compound 6

(GSK8814) the ﬁrst potent, selective, and permeable

B

DOI: 10.1021/acs.jmedchem.8b00862

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ATAD2 chemical probe.⁴⁰Crystal structures of 6 showed that

it also adopts a trans-equatorial conformation when bound to

ATAD2 (Figure 1A) and a trans-triaxial conformation when

bound to BRD4 BD1 (Figure 1B).

substitution vectors similar to the C5′-position of the

piperidine of 6. As a conformationally constrained alternative

to the piperidine, the tropane could direct polar substituents

onto the RVF shelf of ATAD2 and the WPF shelf of BET

without permitting a conformational escape route and so

exploit the diﬀerences between the amino acids there to gain

selectivity.

RESULTS AND DISCUSSION

■

While evaluating the impact of piperidine ring 1,3 sterics on

BET selectivity, we also investigated other conformational

constraints intended to force the shelf substituent into an

equatorial position, including tropane derivatives. The starting

points of this work were small analogues unsubstituted at the

naphthyridone C5-position. As can be seen in Table 2, both

The synthesis of functionalized tropanes is described in

Scheme 1. The C3′-substituent was introduced via an aldol

Scheme 1. Synthetic Route to Functionalized Tropanes and

a

Piperidines

Table 2. Comparison of ATAD2 and BET Activity of

Unfunctionalized Tropane and Piperidines

compd

ATAD2 pIC₅₀

BRD4 BD1 pIC₅₀

7

8

9

10

4.8

4.9

4.3

4.5

5.4

5.5

5.4

5.5

unsubstituted tropane isomers 9 and 10 have similar BET

activity to the piperidine analogue 8. Their potency against

ATAD2 is slightly reduced, in particular for 9, although this

was not thought to be very signiﬁcant.

Crystallographic data (Figure 2) showed that the tropane 10

has a similar ATAD2 binding mode to piperidine compounds

such as 6. We expected that the 3-pyridinyl substituents

previously found to be advantageous at the naphthyridone C5-

position could be incorporated directly into this new template.

We also thought that the tropane could provide access to

a

Reagents and conditions: (a) (Ph)₃P = CHCOOEt, toluene, 100 °C,

75−99%; (b) X = S, NiCl₂, NaBH₄, EtOH, 0 °C, 70%; (c) X = S, m-

CPBA, CH₂Cl₂, 0 °C, 98%; (d) X = CF₂, H₂, Pd/C, MeOH, room

temperature, 98%; (e) LiAlH₄, THF/Et₂O, −30 °C to room

temperature, 88−100%; (f) (COCl)₂, DMSO, CH₂Cl₂, −78 °C

then NEt₃; (g) LDA, THF, −78 °C then 13a, b, or c; (h)

(CH₃SO₂)₂O, NEt₃, CH₂Cl₂, 0 °C; (i) DBU, THF, room temper-

ature, 69−70% over 4 steps; (j) H₂, Pd/C, MeOH/THF, room

temperature, 74−79%; (k) NH₂OH.HCl, pyridine, EtOH, reﬂux, 89−

100%; Na, EtOH, reﬂux, 47−76%.

reaction between ketone 14 and aldehydes 13a−c (readily

available from commercial ketones 11a,b or acid 12c). The

intermediate alcohol was activated as a mesylate and

eliminated to give alkenes 15a,c as single stereoisomers in

good yields. Following a stereoselective hydrogenation of the

double bond, the ketone was converted to the oxime, which,

on reduction with sodium metal, provided racemic amines

16a−c as single diastereoisomers. We did not anticipate that

changing the ether chain (as used in the piperidine series) to

an alkyl chain (tropane series) to access the RVF shelf would

have an impact as it did not have any in the piperidine series

(see pairwise comparison in Figure S1, Supporting Informa-

tion). The racemic synthesis of the analogous C3′−C5′

substituted derivatives 18a−c from commercial 17 has already

been described.³⁹

Figure 2. ATAD2 crystal structures of the NH piperidine GSK8814

(6, white, PDB 5LJ0) and the simpliﬁed tropane 10 (green, PDB

6HDN). Compound 6 uses its piperidine C3′-vector to make its CF₂

interaction with R1077, and its naphthyridone 5-position to make its

pyridyl interaction with D1014. The analogous vectors are available to

the tropane template 10.

These amines were then coupled to the known naphthyr-

idone 19a³⁵(Scheme 2). The derivatives 20 were further

functionalized at the C5 position following bromination and

C

DOI: 10.1021/acs.jmedchem.8b00862

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a

Scheme 2. Synthesis of ATAD2 Inhibitors

a

Reagents and conditions: (a) tBuONa, BrettPhos, Pd₂(dba)₃, THF, 60 °C, 25−94%; (b) TFA, reﬂux, 63−96%; (c) NBS, CH₂Cl₂, 0 °C; (d)

ArB(OH)₂, K₂CO₃, Pd(OAc)₂, di(1-adamantyl)-n-butylphosphine, 1,4-dioxane/water, 100 °C, 71−93% (2 steps); (e) chiral chromatography.

Suzuki coupling. Deprotection in acidic conditions gave the

racemate of inhibitors 22−26. The chiral separation of the

racemic inhibitors or intermediates could be performed at any

stage after the Buchwald coupling. In all cases, one enantiomer

was signiﬁcantly more potent at ATAD2 than the other. Co-

crystallizations of the most active enantiomers in ATAD2 (or

BRD4 BD1/BD2) always showed the same conﬁguration,

which is the one exempliﬁed in all the schemes and tables. The

analogous racemic quinolinone derivatives 27 and 28 (vide

infra) could be obtained using the same sequence, starting

from the known 19b intermediate.³⁴Compound 30 was

obtained by direct deprotection of the corresponding

intermediate 20.

known to be favorable from our previous work, namely the

methylpyridine at the naphthyridone C5-position and the

introduction of tropane substituents intended to interact with

the RVF shelf.^34,35The combination of both modiﬁcations led

to highly potent tropane-containing ATAD2 inhibitors such as

23, 24, and 26, which also show excellent improvements in

BET selectivity. The improvement in potency of these

compounds over 10 is ∼100-fold, which is comparable to

that found relative to 7 in the piperidine series.

Although we were unable to obtain a crystal structure of

ATAD2 bound to 24, we did manage to solve one in complex

with its direct quinolinone N → CH analogue 27. Figure 3

shows ATAD2 bound to 27 superimposed on our previously

published structure of ATAD2 bound to 28, which is the

directly comparable quinolinone analogue of 4.³⁵In our

ATAD2 Activity of Tropane Derivatives. Table 3

compares activities against ATAD2 and BET for the mono-

or disubstituted piperidines and their corresponding tropanes.

These diﬀer from 10 by the incorporation of substituents

Table 3. Comparison of Activities between Tropane and

Piperidine Derivatives

ATAD2

pIC₅₀

BRD4 BD1

pIC₅₀

selectivity

(fold)

compd

X

type

Y

21

22

23

4

N

A

B

C

A

B

C

A

B

C

A

O

6.5

6.7

6.6

6.9

6.6

7.4

7.3

7.0

6.6

6.8

5.0

4.5

5.1

4.8

3.8

<4.3

5.2

4.6

5.3

5.4

30

160

30

125

1260

>250

150

500

60

SO₂

CF₂

SO₂

5

24

25

6

26

a

27

a

CH

20

25

28

Figure 3. Superimposed crystal structures of the ATAD2-bound

racemate of compound 27 (green, PDB 6HD0) and its direct

piperidine analogue 28 (white, PBD 5A83).

a

Compounds are racemic.

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Article

previous work, we saw no structural diﬀerences in binding

mode between the naphthyridone and quinolinone tem-

plates.³⁴The ATAD2 binding mode of the substituted tropane

derivative is very similar to its piperidine counterpart. Both

derivatives form a hydrogen bond to Asp1071 via the

piperidine or tropane nitrogen, anchoring this part of the

molecule to the binding site. In the RVF shelf subpocket, a

sulfone oxygen of each inhibitor is positioned appropriately to

interact with Arg1077. In this particular example, the slight

shift of the shelf substituent seen in the tropane series

compared to the piperidine series might explain the minor

decreases in ATAD2 potency observed for most pairs of

examples made (Table 3, compare 24 to 4 and 5).

The Selectivity of Constrained Tropane Derivatives

over BRD4. From our previous work in the piperidine series,

we understood that polar C3′-substituents (tetrahydropyran,

diﬂuoro-cyclohexyl, and cyclic sulfonamide) were favored on

the RVF shelf of ATAD2 but poorly tolerated on the WPF

shelf of BET.³⁵Crystal structures showed that when these

compounds bind to BRD4 BD1 their piperidine ring ﬂips to

adopt an axial conformation that allows the C3′-substituents to

escape the WPF shelf environment by placing their polar

functionality into solvent. Presumably this involves some

conformational cost. Clearly, it would be impossible for the

tropane derivatives to adopt this axial conformation.

core is not changed by the modiﬁcations. As expected, because

of its constrained tropane ring, the diﬂuorocyclohexane

substituent of 26 is unable to move away from the WPF

shelf of BRD4 unlike that of 6 (Figure 1B). The CF₂group of

26 is forced to sit on the shelf in a region which is unfavorable

due to steric or electrostatic interactions with Trp81. In this

constrained situation, Trp81 responds to binding of 26

through a local rearrangement, adopting several diﬀerent

rotamers where the side chain moves away from the CF₂

group. These alternative conformers are rarely present in

reported BRD4 BD1 structures in the Protein Data Bank

(Figure S2, Supporting Information), but this increased

disorder is apparently preferable to Trp81 remaining next to

the diﬂuorocyclohexane of the ligand. We attribute the lower

BRD4 activity of the sulfone 24 relative to 26 to the adoption

of a similar binding mode leaving the more polar SO₂

functionality in proximity to a rearranged Trp81.

In a further eﬀort to understand better why the trisubstituted

methoxy piperidines are more selective for ATAD2 over BRD4

BD1 than the tropanes, we made some simpliﬁed analogues

lacking any C3′ substituents large enough to be able to occupy

the WPF shelf (Table 4). The C3′−C5′ dimethoxy piperidine

Table 4. Activity and Selectivity of Piperidine Bearing Small

Substituents

The tropanes 23, 24, and 26 have fairly good BRD4

selectivity, comparable to or better than their monosubstituted

piperidine analogues (21, 4, and 25 respectively, Table 3). The

selectivity for ATAD2 over BRD4 is most pronounced for the

sulfone derivative 24 (>250-fold). This is approaching the

selectivity window achieved by the trisubstituted methoxy-

piperidine sulfone 5. However, the THP and diﬂuoro-

cyclohexyl tropanes 23 and 26 did not quite achieve the

level of selectivity reached by their direct methoxy-piperidine

analogues 22 and 6.

We used X-ray crystallography to investigate the reason why

the BRD4 potency of tropane derivative 26 is around 5-fold

greater than the disubstituted piperidine 6. The X-ray structure

of BRD4 BD1 bound to compound 26 is shown in Figure 4

compared to the previously published structure of BRD4 BD1

bound to 1 (PDB 5A85). The position of the naphthyridone

compd

ATAD2 pIC₅₀

BRD4 BD1 pIC₅₀

7

4.8

5.0

4.6

4.5

5.4

5.3

4.6

5.5

29

30

10

is intrinsically less active against BET (compare activity of

compound 30 versus compounds 7, 29, or even 10). The

impact of C3′ and C5′ dimethoxy substitution on ATAD2

potency is much smaller than the impact on BET potency.

This can be understood by examining the crystal structures

of 6 bound to ATAD2 and to BRD4 (Figure 5). The side

chains of the ZA loop are closer to the inhibitor in BRD4 than

ATAD2, leaving less space available at the piperidine C5′

position. The closest side chain of BRD4 to the C5′ atom of 25

in the crystal structure is Leu94 (3.8 Å distance). For

comparison, in ATAD2 the closest side chain the C5′ atom

of 6 is Val1018 (6.1 Å distance). Therefore, we conclude that

in addition to the conformational contribution of the

trisubstituted methoxypiperidine to the selectivity of 6 that

we described previously, there also appears to be a steric

component arising from the more limited amount of accessible

space for the 5′-methoxy group in BRD4.

CONCLUSION

■

We set out to test the idea that constrained bridged piperidine

derivatives would show greater selectivity over the BET

bromodomains by rigidiﬁcation, removing any possibility for

the ligand to avoid placing polar C3′ substituents onto the

WPF shelf. This led to the discovery of compounds such as 24

and 26 that are structurally diﬀerent from our previous series.

Figure 4. X-ray crystallographic binding mode of compound 26

(green, PDB 6HDQ) in BRD4 BD1, showing alternative conformers

of Trp81 (orange). This is superimposed on the BRD4 structure of

the racemate of 1 (white, PDB 5A85).

E

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hoped, for similar reasons to those in our example. The

explanation often remains poorly understood because

crystallography of the antitargets may not be available or

because crystallization of ligands that bind relatively weakly is

challenging. However, when successful the structural insights

may reveal unexpected ways that proteins or ligands can adapt

to minimize unfavorable interactions, and this information can

suggest new strategies to improve selectivity.

EXPERIMENTAL SECTION

Protein Expression, ATAD2 and BET Bromodomain Assays,

and Physicochemical Property Measurement. These were

carried out as described previously.³⁵

■

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 liquid chromatography mass spectrometry

(LCMS) using a Waters ZQ instrument. NMR spectra were recorded

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

Figure 5. X-ray structures of ATAD2 bound to 6 (white, PDB 5LJ0)

and BRD4 BD1 bound to 26 (green, PDB 6HDQ). The magenta

dashed line shows the closest contact between 26 and BRD4 Leu94.

Molecular surfaces of the closest side chains to the C3′-position

(BRD4 Leu94 and ATAD2 Val1018) highlight the relatively greater

space that is available for C3′ substituents in ATAD2 compared to

BRD4.

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.

These retain excellent ATAD2 potency and show good

selectivity over BET.

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

The tropane derivatives were, however, less selective than

their disubstituted piperidine analogues. Our eﬀorts to

understand the reasons for this led us to generate SAR with

simpliﬁed compounds in the latter series, showing that

dimethoxy substitution on the piperidine was intrinsically

detrimental to BET activity. A closer examination of the crystal

structures of this series bound to BRD4 and ATAD2 suggest

that this is because the side chains of the ZA loop of BRD4

allow less space for substitution at the C5′ position. This

further explains the exceptional selectivity seen with 6.

The X-ray structure of the complex of BRD4 bound to 26

showed that the tropane does indeed force the diﬂuorocyclo-

hexyl substituent onto the WPF shelf. However, because this is

electrostatically and/or sterically unfavorable, the crystal

structure also revealed that as a result the protein undergoes

an unexpected ligand-induced conformational change of the

Trp81 side chain. This illustrates the complexity that can arise

in interpreting the SAR of weak protein−ligand interactions

and the diﬃculties that can complicate eﬀorts to improve

selectivity over an antitarget like BRD4. Rational modiﬁcations

designed to introduce particular negative interactions to reduce

binding aﬃnity to the antitarget may be less eﬀective than

expected if the system can ﬁnd ways to evade this interaction.

Some systems, like BRD4, may ﬁnd multiple ways to do this. In

our earlier study on the piperidine series, we showed how the

ligand escaped an unfavorable interaction with BRD4 Trp81 by

adopting an unexpected axial conformational state, which

limited the eﬀectiveness of our rational design strategy until

steps were taken to eliminate the unwanted conformer.³⁹Here,

we have shown that even when this escape route was blocked

by rigidiﬁcation of the ligand, the detrimental eﬀects of the

Trp81 interaction were still limited by an unanticipated

conformational change, in this case, the ability of a side

chain to move by adopting multiple rotameric states.

1

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

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; 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; B = acetonitrile. For both methods, the gradient

employed was (Table 5):

Table 5

time (min)

ﬂow rate (mL/min)

%A

%B

0

1

99

3

1

97

1.5

1.9

2.0

0

100

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 to 1000 AMU; scan time 0.27 s; interscan 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; 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 pH10 with ammonia solution; B = acetonitrile. Run

No doubt there are many cases where rational selectivity

design has been attempted and found to be less successful than

F

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Journal of Medicinal Chemistry

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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

210−350 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; interscan

delay 0.20 s.

palladium on carbon (10% w/w, 50% wet, 1 g), and the resulting

mixture was stirred under an atmosphere of hydrogen (1 bar) for 16

h. The catalyst was removed using a pad of Celite (10 g) and rinsed

with EtOAc. The combined organics were concentrated in vacuo to

give ethyl 2-(4,4-diﬂuorocyclohexyl)acetate (12b) (10.94 g, 98%) as a

colorless oil which was used in the next step without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 4.16 (q, J = 7.1 Hz, 2H),

2.26 (d, J = 7.1 Hz, 2H), 2.16−2.04 (m, 2H), 1.97−1.66 (m, 5H),

1.42−1.31 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H).

Compounds 1−10, 19a,b, 21, 25, and 29 synthesis have already

been published.^33,34,38

2-(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)acetaldehyde

(13a). Step 1: A solution of ethyl 2-(1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)acetate (12a) (6.42 g, 29.1 mmol) in THF (150 mL)

under nitrogen at −30 °C was treated with LiAlH₄(1 M in Et₂O, 29.1

mL, 29.1 mmol), and the resulting mixture was stirred for 1.5 h

between −30 °C and −20 °C. The mixture was then carefully treated

with water (0.960 mL, 1 mL/g LiAlH₄), then with 15% w/w NaOH

in water (0.960 mL), and then with 3 times 0.960 mL of water. The

corresponding mixture was stirred at room temperature for 10 min.

The white precipitate formed was ﬁltered through a 2.5 g pad of

Celite and rinsed with THF. The combined organics were

concentrated in vacuo to give 4-(2-hydroxyethyl)tetrahydro-2H-

thiopyran 1,1-dioxide (5.51 g, 106%) as a colorless oil which was

Ethyl 2-(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)acetate

(12a). Step 1: A solution of dihydro-2H-thiopyran-4(3H)-one

(11.45 g, 99.00 mmol) and ethyl 2-(triphenylphosphoranylidene)-

acetate (36.0 g, 103 mmol) in toluene (300 mL) was stirred at 100 °C

for 8 h. Ethyl 2-(triphenylphosphoranylidene)acetate (4.0 g, 11

mmol) was added, and the mixture was stirred for another 4 h and

then was cooled to room temperature. The solvent was removed in

vacuo, and the residue was triturated with Et₂O. The solid Ph₃PO

formed was ﬁltered oﬀ and rinsed with Et₂O, and the combined

organics were concentrated in vacuo. Puriﬁcation of the residue by

ﬂash chromatography on silica gel (340 g column, 10% GLOBAL

gradient (AcOEt in hexanes)) gave ethyl 2-(dihydro-2H-thiopyran-

1

4(3H)-ylidene)acetate (13.7 g, 75%) as colorless oil. H NMR (400

used in the next step without further puriﬁcation. H NMR (400

MHz, CDCl₃) δ ppm 5.69 (s, 1H), 4.16 (q, J = 7.2 Hz, 2H), 3.21 (m,

2H), 2.82−2.73 (m, 4H), 2.59−2.50 (m, 2H), 1.29 (t, J = 7.2 Hz,

3H). Step 2: A solution of ethyl 2-(dihydro-2H-thiopyran-4(3H)-

ylidene)acetate (7.88 g, 42.3 mmol) in EtOH (160 mL) at 0 °C was

treated with nickel(II) chloride (16.4 g, 127 mmol) then with sodium

borohydride (6.40 g, 169 mmol) Portionwise (1 g every 10 min).

Then 2 h after the ﬁnal addition, the solvent was removed in vacuo

and the residue was partitioned between EtOAc and water. The

biphasic phase was ﬁltered through Celite and the layers were

separated. The aqueous phase was extracted twice with EtOAc, and

the combined organics were washed twice with brine, dried over

MgSO₄, and concentrated in vacuo to give ethyl 2-(tetrahydro-2H-

thiopyran-4-yl)acetate (5.6 g, 70%) as a colorless oil, which was used

MHz, CDCl₃) δ 3.80−3.67 (m, 2H), 3.13−2.93 (m, 4H), 2.15 (m,

2H), 1.97−1.50 (m, 6H). Step 2: A solution of oxalyl chloride (2.27

mL, 26.0 mmol) in CH₂Cl₂(100 mL) at −78 °C under nitrogen was

treated with DMSO (2.83 mL, 39.9 mmol) in CH₂Cl₂(5 mL)

dropwise, and the resulting mixture was stirred at this temperature for

15 min before being treated with 4-(2-hydroxyethyl)tetrahydro-2H-

thiopyran 1,1-dioxide (3.56 g, 20.0 mmol) in CH₂Cl₂(15 mL plus 2

mL rinse). The resulting mixture was stirred for 30 min at this

temperature before being treated with NEt₃(8.35 mL, 59.9 mmol).

The resulting mixture was stirred at this temperature for 30 min then

was warmed to room temperature and stirred for 30 min before being

diluted with CH₂Cl_2.The organic phase was washed with water, and

the aqueous phase was extracted with CH₂Cl₂. The combined

organics were dried using a phase separator and concentrated in vacuo

to give 2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)acetaldehyde

(13a) (3.52 g, 100%) as a white solid which was used in the next

step without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 9.77

(br s, 1H), 3.07−2.99 (m, 4H), 2.52 (dd, J = 6.6, 1.0 Hz, 2H), 2.28−

2.16 (m, 1H), 2.16−2.05 (m, 2H), 1.99−1.81 (m, 2H).

1

in the next step without further puriﬁcation. H NMR (400 MHz,

CDCl₃) δ 4.17 (q, J = 7.1 Hz, 2H), 3.10−2.97 (m, 4H), 2.34 (d, J =

6.6 Hz, 2H), 2.21−2.05 (m, 3H), 2.02−1.85 (m, 2H), 1.28 (t, J = 7.1

Hz, 3H). Step 3: A solution of ethyl 2-(tetrahydro-2H-thiopyran-4-

yl)acetate (5.6 g, 30 mmol) in CH₂Cl₂(150 mL) at 0 °C was treated

with m-CPBA (<77% w/w, 12.7 g, 62.5 mmol) in two portions, and

the resulting mixture was stirred at this temperature. After 30 min,

CH₂Cl₂(150 mL) was added to help stirring. After 6 h, the mixture

was treated with sodium thiosulfate (9.40 g, 59.5 mmol) in water (50

mL). The mixture was vigorously stirred for 10 min, and the layers

were separated. The aqueous phase was extracted with CH₂Cl₂, and

the combined organic phases were washed three times with a

saturated NaHCO₃aqueous solution, dried using a phase separator,

and concentrated in vacuo to give ethyl 2-(1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)acetate (12a) (6.43 g, 98%) as a white solid which was

2-(4,4-Diﬂuorocyclohexyl)acetaldehyde (13b). Step 1: A

solution of ethyl 2-(4,4-diﬂuorocyclohexyl)acetate (12b) (10.5 g,

50.9 mmol) in THF (300 mL) under nitrogen at −30 °C was treated

with LiAlH₄(2 M in Et₂O, 25.5 mL, 51.0 mmol), and the resulting

mixture was stirred for 1.5 h between −30 and −20 °C then was

carefully treated with water (1.9 mL, 1 mL/g LaH) then 1.9 mL of a

15% w/w NaOH in water then with 3 × 1.9 mL of water. The

corresponding mixture was stirred at room temperature for 10 min,

then the white precipitate formed was ﬁltered through Celite (2.5 g

pad) and rinsed with THF. The combined organics were

concentrated in vacuo to give 2-(4,4-diﬂuorocyclohexyl)ethanol

(7.34 g, 88%) as a colorless oil which was used in the next step

1

used in the next step without further puriﬁcation. H NMR (400

MHz, CDCl₃) δ 4.16 (q, J = 7.1 Hz, 2H), 3.10−2.93 (m, 4H), 2.34

(d, J = 6.8 Hz, 2H), 2.19−2.04 (m, 2H), 2.00−1.85 (m, 2H), 1.78−

1.72 (m, 1H), 1.28 (t, J = 7.1 Hz, 3H).

1

without further puriﬁcation. H NMR (400 MHz, CDCl₃) δ 3.77−

Ethyl 2-(4,4-Diﬂuorocyclohexyl)acetate (12b). Step 1: A

solution of 4,4-diﬂuorocyclohexanone (7.35 g, 54.8 mmol) and

ethyl 2-(triphenylphosphoranylidene)acetate (21.0 g, 60.3 mmol) in

toluene (100 mL) was stirred at 100 °C for 16 h then was cooled to

room temperature and concentrated in vacuo. The residue was

triturated with Et₂O and the solid formed (Ph₃PO) removed by

ﬁltration and rinsed with Et₂O. The combined organics were

concentrated in vacuo. Puriﬁcation of the residue obtained by ﬂash

chromatography on silica gel (340 g column, 10% GLOBAL gradient

(AcOEt in hexanes)) gave ethyl 2-(4,4-diﬂuorocyclohexylidene)-

acetate (11.0 g, 99%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)

δ 5.75 (s, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.10−3.01 (m, 2H), 2.46−

2.37 (m, 2H), 2.13−1.96 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H). Step 2: A

solution of ethyl 2-(4,4-diﬂuorocyclohexylidene)acetate (11.0 g, 54.0

mmol) in MeOH (160 mL) at room temperature was treated with

3.63 (m, 2H), 2.15−2.01 (m, 2H), 1.89−1.62 (m, 5H), 1.59−1.46

(m, 3H), 1.39−1.20 (m, 2H). Step 2: A solution of oxalyl chloride

(5.87 mL, 67.1 mmol) in CH₂Cl₂(250 mL) at −78 °C under

nitrogen was treated with DMSO (7.93 mL, 112 mmol) in CH₂Cl₂

(10 mL) dropwise, and the resulting mixture was stirred at this

temperature for 20 min before being treated with 2-(4,4-

diﬂuorocyclohexyl)ethanol (7.34 g, 44.7 mmol) in CH₂Cl₂(20 mL

+ 2 mL rinse). The resulting mixture was stirred for 30 min at this

temperature before being treated with triethylamine (19.9 mL, 143

mmol). The resulting mixture was stirred at this temperature for 1 h,

then was warmed to room temperature, stirred 20 min, and diluted

with CH₂Cl₂. The organic phase was washed with water, and the

aqueous phase was extracted with CH₂Cl₂. The combined organics

were washed with water/brine 1:1, dried using a phase separator, and

concentrated in vacuo to give 2-(4,4-diﬂuorocyclohexyl)acetaldehyde

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(13b) (7.5 g, assumed quantitative) as a yellow solid which was used

in the next step without further puriﬁcation. H NMR (400 MHz,

CDCl₃) δ 9.80 (t, J = 1.6 Hz, 1H), 2.43 (dd, J = 6.8, 1.6, Hz, 2H),

2.17−1.95 (m, 2H), 2.04−1.95 (m, 1H), 1.87−1.78 (m, 3H), 1.78−

1.66 (m, 1H), 1.43−1.29 (m, 2H).

dioxidotetrahydro-2H-thiopyran-4-yl)-1-((methylsulfonyl)oxy)ethyl)-

3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (9.58 g, 100%) as a

white foam which was used in the next step without further

puriﬁcation. Step 3: A solution of crude tert-butyl 2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)-1-((methylsulfonyl)oxy)ethyl)-

3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (9.58 g, 20.0 mmol) in

THF (100 mL) at room temperature was treated with DBU (6.02 mL,

39.9 mmol), and the resulting mixture was stirred at this temperature

for 30 min then most of the solvent was evaporated in vacuo. The

residue was partitioned between EtOAc and water, and the layers

were separated. The aqueous phase was extracted twice with EtOAc,

and the combined organics were washed with brine, dried over

MgSO₄, and concentrated in vacuo to give a yellow solid. Trituration

of this residue with methanol gave (E)-tert-butyl 2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)ethylidene)-3-oxo-8-azabicyclo-

[3.2.1]octane-8-carboxylate (15a) (4.11 g, 54%) as a white solid. The

mother liquors were concentrated in vacuo. Puriﬁcation of the residue

obtained by ﬂash chromatography on silica gel (100 g column, 50%

GLOBAL gradient (AcOEt in hexanes)) gave (E)-tert-butyl 2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)ethylidene)-3-oxo-8-azabicyclo-

[3.2.1]octane-8-carboxylate (15a) (1.18 g, 15%) as a white solid after

trituration with MeOH. Overall yield of 69% over 4 steps. The

stereochemistry of the alkene was conﬁrmed by NMR. LCMS

1

2-(Tetrahydro-2H-pyran-4-yl)acetaldehyde (13c). Step 1: A

solution of 2-(tetrahydro-2H-pyran-4-yl)acetic acid (12c) (5.00 g,

34.7 mmol) in THF (100 mL) at 0 °C under nitrogen was slowly

treated with LiAlH₄(1 M in THF, 34.7 mL, 34.7 mmol). The mixture

was then allowed to warm to room temperature and was stirred for 24

h. The mixture was then cooled to 0 °C and carefully treated with

water (1.3 mL, 1 mL/g LiAlH₄) then with 1.3 mL of 15% w/w NaOH

in water then with 3 × 1.3 mL of water. The resulting mixture was

stirred at room temperature for 10 min, then the white precipitate

formed was ﬁltered through a pad of Celite (2.5 g) and rinsed with

EtOAc. The combined organic phases were concentrated in vacuo.

The residue was dissolved in CH₂Cl₂, and the organic phase was dried

using a phase separator and concentrated in vacuo to give 2-

(tetrahydro-2H-pyran-4-yl)ethanol (4.28 g, 95%) as a very pale-yellow

1

oil which was used in the next step without further puriﬁcation. H

NMR (400 MHz, CDCl₃) δ 3.96 (dd, J = 11.1 4.0 Hz, 2H), 3.78−

3.65 (m, 2H), 3.40 (t, J = 11.7 Hz, 2H), 1.79−1.24 (m, 8H). Step 2:

A solution of oxalyl chloride (4.29 mL, 49.0 mmol) in CH₂Cl₂(100

mL) under nitrogen was cooled to −78 °C then treated with DMSO

(5.8 mL, 82 mmol) in CH₂Cl₂(15 mL) dropwise. The resulting

mixture was stirred for 15 min at this temperature before being

treated with 2-(tetrahydro-2H-pyran-4-yl)ethanol (4.25 g, 32.6

mmol) in CH₂Cl₂(20 mL). The resulting mixture was stirred at

this temperature for 30 min before being treated with NEt₃(14.6 mL,

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

1 h then was warmed to room temperature and diluted with CH₂Cl₂

after 20 min. The organic phase was washed with water (which was

then extracted with CH₂Cl₂), and the combined organics were washed

with water again, dried using a phase separator, and concentrated in

vacuo to give crude 2-(tetrahydro-2H-pyran-4-yl)acetaldehyde (13c)

(5 g, assumed quantitative) as a pale-yellow oil which was used in the

next step without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ

9.79 (t, J = 1.7 Hz, 1H), 3.99−3.92 (m, 2H), 3.44 (dt, J = 11.8, 2.1

Hz, 2H), 2.40 (dd, J = 6.7, 1.8, Hz, 2H), 2.24−2.08 (m, 1H), 1.69−

1.59 (m, 2H), 1.44−1.30 (m, 2H).

(E)-tert-Butyl 2-(2-(1,1-Dioxidotetrahydro-2H-thiopyran-4-

yl)ethylidene)-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate

(15a). Step 1: A solution of diisopropylamine (3.98 mL, 28.0 mmol)

in THF (100 mL) at −78 °C under nitrogen was treated with n-

butyllithium (1.6 M in hexanes, 16.2 mL, 26.0 mmol) dropwise. After

5 min, the mixture was warmed to 0 °C using an ice bath, stirred 30

min at this temperature, and then cooled again at −78 °C. The

mixture was then treated with tert-butyl 3-oxo-8-azabicyclo[3.2.1]-

octane-8-carboxylate (14) (5.40 g, 24.0 mmol) in THF (25 mL plus 5

mL rinse). The resulting mixture was stirred for 1 h at this

temperature before being treated with 2-(1,1-dioxidotetrahydro-2H-

thiopyran-4-yl)acetaldehyde (13a) (3.52 g, 20.0 mmol) in THF (10

mL plus 2 mL rinse). The resulting mixture was stirred for 2 h at −78

°C then was treated with a saturated NH₄Cl aqueous solution. The

mixture was warmed to room temperature and diluted with EtOAc

and water. The layers were separated. The aqueous phase was

extracted with EtOAc, and the combined organics were washed with

brine, dried over MgSO₄, and concentrated in vacuo to give crude

tert-butyl 2-(2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-1-hydrox-

yethyl)-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (9.0 g, 112%)

as a pale-yellow solid which was used in the next step without further

puriﬁcation. Step 2: A solution of crude tert-butyl 2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)-1-hydroxyethyl)-3-oxo-8-

azabicyclo[3.2.1]octane-8-carboxylate (9.0 g, 20 mmol) in CH₂Cl₂

(100 mL) at 0 °C under nitrogen was treated with NEt₃(5.57 mL,

39.9 mmol) then methanesulfonic anhydride (3.83 g, 22.0 mmol) and

the resulting mixture was stirred at this temperature for 40 min and

then was washed with water. The aqueous phase was extracted with

CH₂Cl₂and the combined organics were dried using a phase

separator and concentrated in vacuo to give crude tert-butyl 2-(2-(1,1-

1

(method high pH): Retention time 0.93 min, [M + H]⁺= 384.3. H

NMR (600 MHz, DMSO-d₆) δ 6.36 (t, J = 7.9 Hz, 1H), 5.01 (d, J =

6.0 Hz, 1H), 4.33 (dd, J = 6.0, 4.5 Hz, 1H), 3.15−3.07 (m, 2H),

3.04−2.97 (m, 2H), 2.63 (dd, J = 18.0, 4.5 Hz, 1H), 2.38 (d, J = 18.3

Hz, 1H), 2.25−2.18 (m, 2H), 2.18−2.09 (m, 2H), 1.96 (d, J = 13.9

Hz, 2H), 1.85−1.76 (m, 1H), 1.71−1.61 (m, 3H), 1.61−1.53 (m,

1H), 1.38 (s, 9H).

(E)-tert-Butyl 2-(2-(4,4-Diﬂuorocyclohexyl)ethylidene)-3-

oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (15b). Step 1: A

solution of diisopropylamine (8.92 mL, 62.6 mmol) in THF (200

mL) at −78 °C under nitrogen was treated with n-butyllithium (1.6 M

in hexanes, 36.3 mL, 58.1 mmol) dropwise, and after 5 min the

mixture was warmed to 0 °C using an ice bath, stirred 30 min at this

temperature, then cooled again at −78 °C. The solution was then

treated with tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate

(14) (12.1 g, 53.6 mmol) in THF (25 mL plus 3 mL rinse), and the

resulting mixture was stirred for 1 h at this temperature before being

treated with 2-(4,4-diﬂuorocyclohexyl)acetaldehyde (13b) (7.25 g,

44.7 mmol) in THF (25 mL plus 4 mL rinse). The resulting mixture

was stirred for 2 h at this temperature then was treated with a

saturated NH₄Cl aqueous solution and warmed to room temperature.

The mixture was partitioned between EtOAc and water, and the layers

were separated. The aqueous phase was extracted with EtOAc, and

the combined organics were washed with brine, dried over MgSO₄,

and concentrated in vacuo to give crude tert-butyl 2-(2-(4,4-

diﬂuorocyclohexyl)-1-hydroxyethyl)-3-oxo-8-azabicyclo[3.2.1]octane-

8-carboxylate (20 g, assumed quantitative) as a pale-yellow solid

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

solution of crude tert-butyl 2-(2-(4,4-diﬂuorocyclohexyl)-1-hydrox-

yethyl)-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (20 g, 44.7

mmol) in CH₂Cl₂(200 mL) at 0 °C under nitrogen was treated

with NEt₃(12.5 mL, 89.0 mmol) then methanesulfonic anhydride

(8.57 g, 49.2 mmol) in CH₂Cl₂(40 mL), and the resulting mixture

was stirred at this temperature for 40 min. The organic phase was

then washed with water. The aqueous phase was extracted with

CH₂Cl₂, and the combined organics were washed with brine/water

1:1, dried using a phase separator, and concentrated in vacuo to give

crude tert-butyl 2-(2-(4,4-diﬂuorocyclohexyl)-1-((methylsulfonyl)-

oxy)ethyl)-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (23 g, as-

sumed quantitative) as a brown oil which was used in the next step

without further puriﬁcation. Step 3: A solution of crude tert-butyl 2-

(2-(4,4-diﬂuorocyclohexyl)-1-((methylsulfonyl)oxy)ethyl)-3-oxo-8-

azabicyclo[3.2.1]octane-8-carboxylate (23 g, 44.7 mmol) in THF

(100 mL) at room temperature was treated with DBU (13.5 mL, 89.0

mmol), and the resulting mixture was stirred at this temperature for

30 min then most of the solvent was removed in vacuo. The residue

was partitioned between EtOAc and water and the layers were

H

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Journal of Medicinal Chemistry

Article

separated. The aqueous phase was extracted with EtOAc and the

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

concentrated in vacuo. Puriﬁcation of the residue obtained by ﬂash

chromatography on silica gel (330 g column, 10% GLOBAL gradient

(AcOEt in hexanes)) gave (E)-tert-butyl 2-(2-(4,4-

diﬂuorocyclohexyl)ethylidene)-3-oxo-8-azabicyclo[3.2.1]octane-8-car-

boxylate (17.94 g, 108%) as a pale-yellow oil contaminated with tert-

butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate. This material

was used in the next step without further puriﬁcation. LCMS

warm MeOH (150 mL), and THF (50 mL) was added to facilitate

dissolution. The suspension was then treated with palladium on

carbon (10% w/w, 50% wet, 1 g), and the resulting mixture was

stirred at room temperature under an atmosphere of hydrogen (1 bar)

for 4 h. The catalyst was removed using a pad of Celite (2.5 g) and

rinsed with AcOEt and the combined organics were concentrated in

vacuo to give a white solid. Puriﬁcation of this residue by ﬂash

chromatography on silica gel (25 g column, 50% Global gradient

(AcOEt in hexanes)) gave (1R*,2S*,5S*)-tert-butyl 2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-3-oxo-8-azabicyclo-

[3.2.1]octane-8-carboxylate (4.1 g, 74%) as a white solid. The

stereochemistry of this derivative was conﬁrmed by NMR experi-

1

(method high pH): Retention time 1.29 min, [M + H]⁺= 370.4. H

NMR (400 MHz, CDCl₃) δ 6.56 (s, 1H), 5.17−4.94 (m, 1H), 4.50

(br s, 3H), 2.90−2.56 (m, 3H), 2.47−2.31 (m, 3H), 2.30−1.99 (m,

3H), 1.88−1.58 (m, 4H), 1.45 (s, 9H), 1.41−1.24 (m, 2H).

1

ments. H NMR (400 MHz DMSO-d₆): δ 4.46−4.32 (m, 1H), 4.26

(dd, J = 7.5, 4.0 Hz, 1H), 3.08−2.89 (m, 4H), 2.64 (dd, J = 15.1, 4.5

Hz, 1H), 2.49−2.43 (m, 1H), 2.20 (dd, J = 15.0, 1.9 Hz, 1H), 2.03 (s,

2H), 2.01−1.91 (m, 1H), 1.92−1.82 (m, 1H), 1.90−1.81 (m, 1H),

1.74−1.62 (m, 2H), 1.67−1.55 (m, 1H), 1.57−1.51 (m, 1H), 1.61−

1.50 (m, 1H), 1.49 (s, 9H), 1.40−1.29 (m, 2H),1.08 (ddt, J = 14.0,

9.0, 7.0 Hz, 1H). Step 2: A solution of (1R*,2S*,5S*)-tert-butyl 2-(2-

(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-3-oxo-8-azabicyclo-

[3.2.1]octane-8-carboxylate (0.450 g, 1.17 mmol) in EtOH (20 mL)

at room temperature was treated with hydroxylamine hydrochloride

(0.162 g, 2.33 mmol) and pyridine (0.189 mL, 2.33 mmol), and the

resulting mixture was stirred at reﬂux for 1 h then was 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 phase separator and

concentrated in vacuo to give (1R*,2S*,5S*)-tert-butyl 2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-3-(hydroxyimino)-8-

azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 107%, mixture of Z and

E isomers) as a colorless oil which was used in the next step without

(E)-tert-Butyl-3-oxo-2-(2-(tetrahydro-2H-pyran-4-yl)-

ethylidene)-8-azabicyclo[3.2.1]octane-8-carboxylate (15c).

Step 1: A solution of diisopropylamine (6.50 mL, 45.6 mmol) in

THF (150 mL) at −78 °C under nitrogen was treated with n-

butyllithium (1.6 M in hexanes, 26.5 mL, 42.4 mmol), and after 5

min, the resulting mixture was warmed to 0 °C using an ice bath and

stirred at this temperature for 30 min before being cooled again to

−78 °C. The mixture was then treated with tert-butyl 3-oxo-8-

azabicyclo[3.2.1]octane-8-carboxylate (14) (8.81 g, 39.1 mmol) (in

20 mL of THF) dropwise, and the mixture was stirred 90 min before

being treated with crude 2-(tetrahydro-2H-pyran-4-yl)acetaldehyde

(13c) (4.18 g, 32.6 mmol) in 20 mL of THF. The resulting yellow

solution was stirred for 2 h at this temperature before a saturated

NH₄Cl aqueous solution (100 mL) was added at −78 °C. The

mixture was warmed to room temperature and diluted with EtOAc

and water. The layers were separated, and the aqueous phase was

extracted twice with EtOAc. The combined organics were washed

with brine, dried over MgSO₄, and concentrated in vacuo to give 14 g

of crude tert-butyl 2-(1-hydroxy-2-(tetrahydro-2H-pyran-4-yl)ethyl)-

3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate as a brown oil which

was used in the next step without further puriﬁcation. Assumed

quantitative. Step 2: A solution of crude tert-butyl 2-(1-hydroxy-2-

(tetrahydro-2H-pyran-4-yl)ethyl)-3-oxo-8-azabicyclo[3.2.1]octane-8-

carboxylate (14.0 g, 32,6 mmol) in CH₂Cl₂(200 mL) at 0 °C under

nitrogen was treated with NEt₃(9.09 mL, 65.2 mmol) then with

methanesulfonic anhydride (5.96 g, 34.2 mmol) in CH₂Cl₂(20 mL).

The resulting pale-yellow mixture was stirred at this temperature for 1

h then was diluted with CH₂Cl₂and washed with water. The aqueous

phase was extracted with CH₂Cl_2.The combined organics were dried

using a phase separator and concentrated in vacuo to give crude tert-

butyl 2-(1-((methylsulfonyl)oxy)-2-(tetrahydro-2H-pyran-4-yl)-

ethyl)-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (15.0 g, consid-

ered quantitative) as a light-brown oil which was used in the next step

without further puriﬁcation. Step 3: A solution of crude tert-butyl 2-

(1-((methylsulfonyl)oxy)-2-(tetrahydro-2H-pyran-4-yl)ethyl)-3-oxo-

8-azabicyclo[3.2.1]octane-8-carboxylate (15.0 g, 32.6 mmol) in THF

(200 mL) at room temperature was treated with DBU (9.83 mL, 65.2

mmol), and the resulting yellow-brown mixture was stirred at this

temperature for 45 min then most of the THF was removed in vacuo.

The residue was partitioned between EtOAc and water, and the layers

were separated. The aqueous phase was extracted with EtOAc, and

the combined organics were washed with brine, dried over MgSO₄,

and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (330 G column, 40% GLOBAL gradient

(AcOEt in hexanes)) gave (E)-tert-butyl 3-oxo-2-(2-(tetrahydro-2H-

pyran-4-yl)ethylidene)-8-azabicyclo[3.2.1]octane-8-carboxylate (15c)

(7.6 g, 70% yield over 4 steps) as a colorless oil. LCMS (method high

1

further puriﬁcation. H NMR (400 MHz, CDCl₃) δ 7.60−7.39 (m,

1H), 4.42−4.07 (m, 2H), 3.24−3.14 (m, 1H), 3.05 (br s, 2H), 2.96

(br s, 2H), 2.61−2.33 (m, 1H), 2.11 (br s, 2H), 2.10−1.65 (m, 8H),

1.60−1.36 (m, 12H), 1.25−1.11 (m, 1H). Step 3: A solution of

(1R*,2S*,5S*)-tert-butyl 2-(2-(1,1-dioxidotetrahydro-2H-thiopyran-

4-yl)ethyl)-3-(hydroxyimino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(0.30 g, 0.75 mmol) in EtOH (20 mL) at reﬂux under nitrogen was

treated with sodium (1 g) portionwise (around 50 mg each) over 1 h,

allowing eﬀervescence to cease before adding the next piece. The

mixture was then cooled to room temperature and concentrated in

vacuo. The residue was partitioned between water and EtOAc, and

the layers were separated. The organic phase was dried over MgSO₄

and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

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

NH₃in MeOH) in CH₂Cl₂) gave (1R*,2R*,3S*,5S*)-tert-butyl 3-

amino-2-(2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-8-

azabicyclo[3.2.1]octane-8-carboxylate (16a) (0.22 g, 76%) as a pale-

yellow gum. ¹H NMR (600 MHz, CDCl₃) δ 4.33−3.94 (m, 1H), 3.05

(br s, 1H), 2.97 (d, J = 13.6 Hz, 1H), 2.71−2.62 (m, 1H), 2.14 (br s,

2H), 2.03−1.95 (m, 1H), 1.89−1.72 (m, 7H), 1.70−1.19 (m, 19H),

1.12−1.02 (m, 1H).

(1R*,2R*,3S*,5S*)-tert-Butyl 3-Amino-2-(2-(4,4-

diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octane-8-carbox-

ylate (16b). Step 1: A solution of (E)-tert-butyl 2-(2-(4,4-

diﬂuorocyclohexyl)ethylidene)-3-oxo-8-azabicyclo[3.2.1]octane-8-car-

boxylate (15b) (14.5 g, 39.2 mmol) in MeOH (250 mL) was treated

with palladium on carbon (10% w/w, 50% wet, 2 g), and the resulting

mixture was stirred under an atmosphere of hydrogen for 16 h. The

catalyst was ﬁltered oﬀ using a pad of Celite (10 g) and rinsed with

EtOAc and the combined organics were concentrated in vacuo.

Puriﬁcation of this residue by ﬂash chromatography on silica gel (340

g column, 20% Global gradient (AcOEt in hexanes)) gave

(1R*,2S*,5S*)-tert-butyl 2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-3-oxo-8-

azabicyclo[3.2.1]octane-8-carboxylate (7.07 g, 48%) as a white solid.

¹H NMR (600 MHz, DMSO-d6) δ 4.34−4.29 (m, 1H), 4.24−4.11

1

pH): Retention time 1.09 min, [M + H]⁺= 336.4. H NMR (400

MHz, CDCl₃) δ 6.63−6.51 (m, 1H), 5.18−4.96 (m, 1H), 4.58−4.40

(m, 1H), 3.97 (dt, J = 11.5, 2.0 Hz, 2H), 3.43−3.30 (m, 2H), 2.91−

2.62 (m, 1H), 2.41 (dd, J = 18.1, 1.0 Hz, 1H), 2.31−2.04 (m, 4H),

1.77−1.58 (m, 5H), 1.47 (s, 9H), 1.42−1.26 (m, 2H).

(1R*,2R*,3S*,5S*)-tert-Butyl 3-Amino-2-(2-(1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-

8-carboxylate (16a). Step 1: (E)-tert-Butyl 2-(2-(1,1-dioxidotetra-

hydro-2H-thiopyran-4-yl)ethylidene)-3-oxo-8-azabicyclo[3.2.1]-

octane-8-carboxylate (15a) (5.5 g, 14.34 mmol) was suspended in

(m, 1H), 2.68−2.52 (m, 1H), 2.46−2.38 (m, 1H), 2.17 (d, J = 14.7

Hz, 1H), 2.03−1.93 (m, 2H), 1.96−1.89 (m, 1H), 1.91−1.80 (m,

1H), 1.85−1.78 (m, 1H), 1.80−1.69 (m, 4H), 1.53−1.45 (m, 2H),

1.44 (s, 9H), 1.38−1.29 (m, 1H), 1.26−1.20 (m, 2H),1.19−1.07 (m,

I

DOI: 10.1021/acs.jmedchem.8b00862

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Journal of Medicinal Chemistry

Article

2H), 1.06−0.98 (m, 1H). Step 2: A solution of (1R*,2S*,5S*)-tert-

butyl 2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-3-oxo-8-azabicyclo[3.2.1]-

octane-8-carboxylate (3.5 g, 9.4 mmol) in EtOH (20 mL) at room

temperature was treated with hydroxylamine hydrochloride (1.31 g,

18.8 mmol) and pyridine (1.52 mL, 18.8 mmol), and the resulting

mixture was stirred at reﬂux for 1 h then was cooled to room

temperature and diluted with water (40 mL). The mixture was stirred

for 30 min, then the precipitate formed was ﬁltered oﬀ and dried

under vacuum at 40 °C to give (1R*,2S*,5S*)-tert-butyl 2-(2-(4,4-

diﬂuorocyclohexyl)ethyl)-3-(hydroxyimino)-8-azabicyclo[3.2.1]-

octane-8-carboxylate (3.35 g, 92%, mixture of Z and E isomers) which

was used in the next step without further puriﬁcation. ¹H NMR (400

MHz, CDCl₃) δ 7.65−7.42 (m, 1H), 4.44−4.08 (m, 2H), 3.24−3.09

(m, 1H), 2.65−2.36 (m, 1H), 2.06 (br s, 4H), 1.80 (d, J = 12.5 Hz,

4H), 1.72−1.57 (m, 7H), 1.50 (s, 9H), 1.42−1.15 (m, 3H). Step 3: A

solution of (1R*,2S*,5S*)-tert-butyl 2-(2-(4,4-diﬂuorocyclohexyl)-

ethyl)-3-(hydroxyimino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(1.0 g, 2.6 mmol) in EtOH (60 mL) at reﬂux under nitrogen was

treated with sodium (1 g) in small portions (around 50 mg each) over

1 h, allowing eﬀervescence to cease before adding the next piece. The

mixture was then cooled to room temperature and concentrated in

vacuo. The residue was partitioned between water and EtOAc, and

the layers were separated. The organic phase was dried over MgSO₄

and concentrated in vacuo. Puriﬁcation of the residue by ﬂash

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

NH₃in MeOH) in CH₂Cl₂) gave (1R*,2R*,3S*,5S*)-tert-butyl 3-

amino-2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octane-

hydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

(0.40 g, 1.1 mmol) in EtOH (30 mL) at reﬂux. The mixture was

stirred for a further 30 min, then was cooled to room temperature and

diluted with water (100 mL). The mixture was extracted with EtOAc

(2 × 50 mL), and the combined organics were dried over MgSO₄and

concentrated in vacuo. Puriﬁcation of the residue by ﬂash

chromatography on silica gel (gradient: 0−10% (2 M NH₃in

MeOH) in CH₂Cl₂) gave (1R*,2R*,3S*,5S*)-tert-butyl 3-amino-2-(2-

(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carbox-

1

ylate (16) (0.22 g, 57%) as a pale-yellow oil. H NMR (400 MHz,

CDCl₃) δ 4.37−4.05 (m, 2H), 3.99 (dd, J = 11.0, 3.0 Hz, 2H), 3.41

(d, J = 2.0 Hz, 2H), 2.80−2.66 (m, 1H), 2.20−1.93 (m, 3H), 1.83 (d,

J = 3.3 Hz, 3H), 1.75−1.55 (m, 5H), 1.51 (s, 9H), 1.48−1.22 (m,

5H), 1.14−1.01 (m, 2H).

(3R,4r,5S)-tert-Butyl 4-Amino-3,5-dimethoxypiperidine-1-

carboxylate (18a). Step 1: A solution of (3R*,4S*,5S*)-tert-butyl

4-azido-3-hydroxy-5-methoxypiperidine-1-carboxylate (see ref 38, 560

mg, 2.06 mmol) in DMF (8 mL) at 0 °C under nitrogen was treated

with sodium hydride (60% w/w in mineral oil, 99 mg, 2.5 mmol) and

then after 5 min with methyl iodide (0.257 mL, 4.11 mmol). The

resulting mixture was stirred for 30 min at this temperature then was

diluted with water. The aqueous phase was extracted three times with

Et₂O. The combined organics were washed with brine, dried over

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

chromatography on silica gel (25 g column, 20% Global gradient

(AcOEt in hexanes)) gave (3R,4r,5S)-tert-butyl 4-azido-3,5-dimethox-

1

ypiperidine-1-carboxylate (530 mg, 90%) as a colorless oil. H NMR

1

(400 MHz, CDCl₃) δ 4.55−4.17 (m, 2H), 3.51 (s, 6H), 3.31−3.22

(m, 1H), 3.11−2.94 (m, 2H), 2.56−2.37 (m, 2H), 1.49 (s, 9H). Step

2: A solution of (3R,4r,5S)-tert-butyl 4-azido-3,5-dimethoxypiper-

idine-1-carboxylate (500 mg, 1.75 mmol) in MeOH (30 mL) at room

temperature was treated with palladium on carbon (10% w/w, 50%

wet, 100 mg), and the resulting mixture was stirred at room

temperature for 16 h under hydrogen (1 bar). The catalyst was

ﬁltered oﬀ using a pad of Celite (2.5 g) and rinsed with MeOH. The

combined organics were concentrated in vacuo to give (3R,4r,5S)-tert-

butyl 4-amino-3,5-dimethoxypiperidine-1-carboxylate (18a) (410 mg,

90%) as a black solid which was used in the next step without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 4.55−4.14 (m, 2H), 3.46

(s, 6H), 3.02−2.86 (m, 2H), 2.75−2.67 (m, 1H), 2.51−2.32 (m, 2H),

1.81 (br s, 2H), 1.48 (s, 9H).

(3S*,4R*,5R*)-tert-Butyl 4-amino-3-methoxy-5-((tetrahy-

dro-2H-pyran-4-yl)methoxy)piperidine-1-carboxylate (18b).

Step 1: A solution of (tetrahydro-2H-pyran-4-yl)methanol (1.50 g,

12.9 mmol) and pyridine (1.04 mL, 12.9 mmol) in CH₂Cl₂(30 mL)

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

with Tf₂O (2.18 mL, 12.9 mmol) dropwise. The resulting mixture was

stirred for 1 h at 0 °C, then was washed with water (2 × 30 mL),

dried using a phase separator, and concentrated in vacuo to give

(tetrahydro-2H-pyran-4-yl)methyl triﬂuoromethanesulfonate (3.02 g,

94%) as a colorless oil which was used immediately in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 4.38 (d, J

= 6.6 Hz, 2H), 4.08−3.98 (m, 2H), 3.42 (td, J = 11.9, 2.0 Hz, 2H),

2.19−2.06 (m, 1H), 1.74−1.64 (m, 2H), 1.50−1.35 (m, 2H). Step 2:

A solution of (3R*,4S*,5S*)-tert-butyl 4-azido-3-hydroxy-5-methox-

ypiperidine-1-carboxylate (0.82 g, 3.0 mmol) in THF (20 mL) under

nitrogen was cooled to 0 °C using an ice bath, then was treated with

potassium tert-butoxide (0.439 g, 3.91 mmol). The resulting mixture

was stirred at this temperature for 20 min then was treated with the

dropwise addition of (tetrahydro-2H-pyran-4-yl)methyl triﬂuorome-

thanesulfonate (1.12 g, 4.52 mmol). The resulting mixture was stirred

for 1 h at 0 °C, then allowed to warm to room temperature over 1 h

before being diluted with water (60 mL). The aqueous phase was

extracted with EtOAc (2 × 50 mL), and the combined organics were

washed with brine, dried over MgSO₄, and concentrated in vacuo.

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

column, gradient: 0−50% EtOAc in cyclohexane) gave

(3S*,4R*,5R*)-tert-butyl 4-azido-3-methoxy-5-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidine-1-carboxylate (0.74 g, 66%) as a

pale-yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 4.53−4.13 (m,

8-carboxylate (16b) (0.45 g, 47%) as a pale-yellow gum. H NMR

(400 MHz, DMSO-d₆) δ ppm 4.05−3.98 (m, 1H), 3.96−3.84 (m,

1H), 2.55−2.43 (m, 4H), 2.00 (d, J = 7.1 Hz, 2H), 1.89−1.58 (m,

8H), 1.56−1.23 (m, 4H), 1.45 (s, 9H), 1.16 (d, J = 10.6 Hz, 4H),

0.97−0.84 (m, 1H).

(1R*,2R*,3S*,5S*)-tert-Butyl 3-Amino-2-(2-(tetrahydro-2H-

pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

(16c). Step 1: A solution of (E)-tert-butyl 3-oxo-2-(2-(tetrahydro-2H-

pyran-4-yl)ethylidene)-8-azabicyclo[3.2.1]octane-8-carboxylate (15c)

(7.56 g, 22.5 mmol) in MeOH (150 mL) was treated with palladium

on carbon (10% w/w, 50% wet, 1.5 g), and the resulting mixture was

stirred under hydrogen (1 bar) at room temperature for 3 h. The

catalyst was ﬁltered oﬀ through a pad of Celite (2.5 g) and rinsed with

MeOH. The combined organics were concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel

(100 g column, 30% GLOBAL gradient (AcOEt in hexanes)) gave

(1R*,2S*,5S*)-tert-butyl 3-oxo-2-(2-(tetrahydro-2H-pyran-4-yl)-

ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (5.97 g, 79%) as a

1

white foam. H NMR (600 MHz, DMSO-d₆) δ 4.34−4.28 (m, 1H),

4.24−4.13 (m, 1H), 3.85−3.78 (m, 2H), 3.28−3.22 (m, 2H), 2.68−

2.54 (m, 1H), 2.46−2.37 (m, 1H), 2.17 (d, J = 15.0 Hz, 1H), 2.00−

1.89 (m, 1H), 1.87−1.75 (m, 2H), 1.56 (d, J = 12.8 Hz, 2H), 1.51−

1.44 (m, 3H), 1.45 (s, 9H), 1.25−1.19 (m, 2H), 1.18−1.07 (m, 2H),

1.06−0.98 (m, 1H). Step 2: A solution of (1R*,2S*,5S*)-tert-butyl 3-

oxo-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-

8-carboxylate (0.45 g, 1.3 mmol) in EtOH (20 mL) at room

temperature was treated with hydroxylamine hydrochloride (0.185 g,

2.67 mmol) and pyridine (0.216 mL, 2.67 mmol). The resulting

mixture was stirred at reﬂux for 1 h then was 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 phase separator and

concentrated in vacuo to give (1R*,2S*,5S*)-tert-butyl 3-(hydrox-

yimino)-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]-

octane-8-carboxylate (0.42 g, 89%) as a colorless oil (mixture of Z and

E isomers) which was used in the next step without further

1

puriﬁcation. H NMR (400 MHz, CDCl₃) δ 8.97−8.69 (m, 1H),

4.44−4.09 (m, 2H), 4.02−3.84 (m, 2H), 3.37 (t, J = 11.7 Hz, 2H),

3.24−3.08 (m, 1H), 2.66−2.34 (m, 1H), 1.95 (br s, 5H), 1.61 (d, J =

13.0 Hz, 3H), 1.49 (s, 9H), 1.12−1.51 (m, 6H). Step 3: Sodium (1.0

g, 43 mmol) was cut into small pieces and placed in a beaker

containing cyclohexane, then was added piece by piece over 30 min to

a solution of (1R*,2S*,5S*)-tert-butyl 3-(hydroxyimino)-2-(2-(tetra-

J

DOI: 10.1021/acs.jmedchem.8b00862

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Journal of Medicinal Chemistry

Article

2H), 3.99 (dt, J = 11.5, 2.1 Hz, 2H), 3.51 (s, 3H), 3.46−3.34 (m,

3H), 3.31−3.22 (m, 1H), 3.16−3.06 (m, 1H), 2.91−3.04 (m, 1H),

2.58−2.37 (m, 2H), 1.94−1.78 (m, 1H), 1.75−1.59 (m, 3H), 1.48 (s,

9H), 1.42−1.26 (m, 2H). Step 3: A solution of (3S*,4R*,5R*)-tert-

butyl 4-azido-3-methoxy-5-((tetrahydro-2H-pyran-4-yl)methoxy)-

piperidine-1-carboxylate (0.800 g, 2.16 mmol) in MeOH (60 mL)

was hydrogenated in the H Cube over a Pd/C catalyst cartridge at

atmospheric pressure. The eluant was evaporated in vacuo to give

(3S*,4R*,5R*)-tert-butyl 4-amino-3-methoxy-5-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidine-1-carboxylate (18b) (0.72 g, 97%) as

a colorless oil, which was used in the next step without puriﬁcation.

¹H NMR (400 MHz, CDCl₃) δ 4.53−4.19 (br s, 2H), 3.58−3.44 (m,

stirred under microwave irradiations at 100 °C for 30 min then was

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 to give a brown residue.

This was dissolved in TFA (2 mL), reluxed for 4 h, then cooled to

room temperature and concentrated in vacuo. Puriﬁcation of the

residue by MDAP (method high pH) gave 8-(((3S*,4R*,5R*)-3-

methoxy-5-((tetrahydro-2H-pyran-4-yl)methoxy)piperidin-4-yl)-

amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-

one (58 mg, 53%) as a white foam. LCMS (method high pH):

1

Retention time 0.69 min, [M + H]⁺= 494.5. H NMR (400 MHz,

CDCl₃) δ ppm 12.93−13.92 (m, 1H), 8.52 (d, J = 1.5 Hz, 1H), 8.47

(d, J = 2.0 Hz, 1H), 7.96 (s, 1H), 7.75 (d, J = 1.0 Hz, 1H), 7.52 (d, J =

1.0 Hz, 1H), 6.60−6.81 (m, 1H), 4.54−4.73 (m, 1H), 3.77 (br dd, J =

11.2, 2.4 Hz, 1H), 3.48 (br d, J = 3.9 Hz, 3H), 3.45 (s, 4H), 3.34−

3.40 (m, 1H), 3.33−3.42 (m, 1H), 3.25 (dd, J = 8.8, 7.3 Hz, 1H),

3.17−3.22 (m, 1H), 3.11−3.16 (m, 1H), 2.59−2.69 (m, 2H), 2.46 (s,

3H), 2.36 (d, J = 1.0 Hz, 3H), 1.67 (br s, 3H), 1.37 (br dd, J = 13.0,

1.7 Hz, 1H), 1.24−1.32 (m, 1H), 1.11 (qd, J = 12.4, 4.4 Hz, 1H). The

two enantiomers of 8-(((3S*,4R*,5R*)-3-methoxy-5-((tetrahydro-

2H-pyran-4-yl)methoxy)piperidin-4-yl)amino)-3-methyl-5-(5-methyl-

pyridin-3-yl)-1,7-naphthyridin-2(1H)-one were separated by chiral

chromatograhy. Analytical method: Approximately 0.5 mg of

racemate were dissolved in 50% EtOH in heptane (1 mL) and 20

μL were injected onto the column, eluting with 30% EtOH (+0.2%

isopropylamine) in heptane, f = 1.0 mL/min, wavelength 215 nm.

Column 4.6 mm i.d. × 25 cm Chiralcel OD-H. Preparative method:

approximately 44 mg of material were dissolved in 3 mL of EtOH

(+0.2% isopropylamine). Injections (3 in total): 1 mL of the solution

was injected onto the column eluting with 30% EtOH (+0.2%

isopropylamine) in heptane, f = 25 mL/min, wavelength 215 nm.

Column 30 mm × 25 cm Chiralcel OD-H. The appropriate fractions

were combined and concentrated in vacuo to give 8-(((3S,4R,5R)-3-

methoxy-5-((tetrahydro-2H-pyran-4-yl)methoxy)piperidin-4-yl)-

amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-

one (13 mg, 59%) as the fastest running enantiomer and 8-

(((3R,4S,5S)-3-methoxy-5-((tetrahydro-2H-pyran-4-yl)methoxy)-

piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naph-

thyridin-2(1H)-one (11 mg, 50%) as the slowest running enantiomer.

3-Methyl-5-(5-methylpyridin-3-yl)-8-(((1S,2R,3R,5R)-2-(2-

(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octan-3-

yl)amino)-1,7-naphthyridin-2(1H)-one (23). Step 1: A mixture of

(1R*,2R*,3S*,5S*)-tert-butyl 3-amino-2-(2-(tetrahydro-2H-pyran-4-

yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (16c) (0.18 g, 0.53

mmol), 2-(benzyloxy)-8-chloro-3-methyl-1,7-naphthyridine (19a)

(0.15 g, 0.53 mmol), sodium tert-butoxide (0.152 g, 1.58 mmol),

BrettPhos (0.028 g, 0.053 mmol), and Pd₂(dba)₃(0.024 g, 0.026

mmol) in THF (2 mL) was stirred at room temperature for 1 h, then

at 60 °C for 18 h. The mixture was then cooled to room temperature

and diluted with EtOAc (20 mL). The organic phases 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 0−80% EtOAc in cyclohexane) gave (1S*,2S*,3R*,5R*)-tert-

butyl 3-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-2-(2-

(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carbox-

ylate (0.245 g, 79%). LCMS (method high pH): Retention time 1.08

2H), 3,51 (s, 3H), 3.33−3.21 (m, 1H), 3.16−3.06 (m, 3H), 3.05−

2.93 (m, 3H), 2.59−2.39 (m, 2H), 2.28−2.15 (m, 2H), 2.00−1.80

(m, 3H), 1.51−1.42 (m, 2H), 1.48 (s, 9H).

8-(((3S,4R,5R)-3-Methoxy-5-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidin-4-yl)amino)-3-methyl-5-(5-methylpyridin-

3-yl)-1,7-naphthyridin-2(1H)-one (22). Step 1: A mixture of

(3S*,4R*,5R*)-tert-butyl 4-amino-3-methoxy-5-((tetrahydro-2H-

pyran-4-yl)methoxy)piperidine-1-carboxylate (18b) (0.200 g, 0.579

mmol), 2-(benzyloxy)-8-chloro-3-methyl-1,7-naphthyridine (19a)

(0.15 g, 0.53 mmol), sodium tert-butoxide (0.152 g, 1.58 mmol),

BrettPhos (0.028 g, 0.053 mmol), and Pd₂(dba)₃(0.024 g, 0.026

mmol) in THF (2 mL) was stirred at room temperature for 1 h, then

at 60 °C for 18 h before being cooled to room temperature and

diluted with EtOAc (20 mL). 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: 0−100% EtOAc in cyclohexane) gave (3S*,4R*,5R*)-tert-

butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-3-

methoxy-5-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carbox-

ylate (0.12 g, 38%) as a pale-yellow gum. LCMS (method high pH):

1

Retention time 1.42 min, [M + H]⁺= 593. H NMR (400 MHz,

CDCl₃) δ 7.92 (d, J = 5.9 Hz, 1H), 7.68 (s, 1H), 7.55−7.47 (m, 2H),

7.45−7.38 (m, 2H), 7.35 (d, J = 7.1 Hz, 1H), 6.73 (d, J = 5.6 Hz,

1H), 6.25 (d, J = 9.0 Hz, 1H), 5.52 (s, 2H), 4.58−4.24 (m, 2H),

3.69−3.31 (m, 9H), 3.08−2.86 (m, 3H), 2.83−2.63 (m, 2H), 2.41 (s,

3H), 1.54 (s, 9H), 1.45−1.34 (m, 1H), 1.29−1.16 (m, 1H), 1.14−

1.03 (m, 1H), 1.01−0.87 (m, 1H), 0.80−0.64 (m, 1H). Step 2: A

solution of (3S*,4R*,5R*)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-

naphthyridin-8-yl)amino)-3-methoxy-5-((tetrahydro-2H-pyran-4-yl)-

methoxy)piperidine-1-carboxylate (130 mg, 0.219 mmol) in CH₂Cl₂

(10 mL) was cooled using an ice bath then was treated with NBS

(39.0 mg, 0.219 mmol), and the resulting mixture was stirred at this

temperature for 1 h. The mixture was then treated with a saturated

aqueous sodium metabisulphite solution, and after 10 min the layers

were separated. The organic phase was dried using a phase separator

and concentrated in vacuo to give (3S*,4R*,5R*)-tert-butyl 4-((2-

(benzyloxy)-5-bromo-3-methyl-1,7-naphthyridin-8-yl)amino)-3-me-

thoxy-5-((tetrahydro-2H-pyran-4-yl)methoxy)piperidine-1-carboxy-

late (0.15 g, 102%) which was used in the next step without further

puriﬁcation.

LCMS (method high pH): Retention time 1.56 min, [M + H]⁺=

673.6 (1 Br). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.05 (s, 1H), 7.99

(d, J = 1.0 Hz, 1H), 7.50 (d, J = 6.8 Hz, 2H), 7.39−7.44 (m, 2H),

7.33−7.38 (m, 1H), 6.26 (d, J = 9.3 Hz, 1H), 5.52 (s, 2H), 4.17−4.57

(m, 2H), 4.05−4.16 (m, 1H), 3.59−3.73 (m, 2H), 3.43−3.59 (m,

2H), 3.38 (s, 3H), 3.32−3.37 (m, 1H), 2.94−3.11 (m, 3H), 2.60−

2.78 (m, 2H), 2.47 (d, J = 1.0 Hz, 3H), 1.54 (s, 9H), 1.39−1.50 (m,

1H), 1.21−1.30 (m, 1H), 1.08−1.17 (m, 1H), 0.98 (qd, J = 12.3, 4.6

Hz, 1H), 0.68−0.82 (m, 1H). Step 3: A 20 mL microwave vial was

charged with (5-methylpyridin-3-yl)boronic acid (0.046 g, 0.335

mmol), (3S*,4R*,5R*)-tert-butyl 4-((2-(benzyloxy)-5-bromo-3-meth-

yl-1,7-naphthyridin-8-yl)amino)-3-methoxy-5-((tetrahydro-2H-pyran-

4-yl)methoxy)piperidine-1-carboxylate (0.150 g, 0.223 mmol),

K₂CO₃(0.093 g, 0.67 mmol), Pd(OAc)₂(5.0 mg, 0.022 mmol),

di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cataCXium A) (8.0

mg, 0.022 mmol), and then was ﬁlled with 1,4-dioxane (4 mL), and

water (2 mL). The reaction mixture was stirred and degassed for 20

min with nitrogen. The reaction vessel was sealed and the mixture was

1

min, [M + H]⁺= 587.6. H NMR (400 MHz, CDCl₃) δ 7.86 (d, J =

5.6 Hz, 1H), 7.65 (d, J = 1.0 Hz, 1H), 7.52−7.46 (m, 2H), 7.45−7.39

(m, 2H), 7.37−7.30 (m, 1H), 6.70 (d, J = 5.9 Hz, 1H), 5.92 (d, J =

9.0 Hz, 1H), 5.49 (s, 2H), 4.30 (br s, 3H), 3.87 (dd, J = 11.0, 2.7 Hz,

2H), 3.34−3.21 (m, 2H), 2.79 (s, 1H), 2.45 (d, J = 1.0 Hz, 3H),

2.15−1.99 (m, 2H), 1.99−1.80 (m, 3H), 1.79−1.43 (m, 14H), 1.37

(d, J = 10.5 Hz, 2H), 1.29−1.08 (m, 3H). Step 2: A solution of

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-3-methyl-1,7-naph-

thyridin-8-yl)amino)-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-8-

azabicyclo[3.2.1]octane-8-carboxylate (0.18 g, 0.31 mmol) in CH₂Cl₂

(10 mL) at 0 °C was treated with NBS (0.055 g, 0.31 mmol), and the

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

treated with a 10% w/w sodium metabisulphite aqueous solution. The

K

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biphasic mixture was stirred for 20 min, and then the layers were

separated. The organic layer was dried using an hydrophobic frit and

concentrated in vacuo to give (1S*,2S*,3R*,5R*)-tert-butyl 3-((2-

(benzyloxy)-5-bromo-3-methyl-1,7-naphthyridin-8-yl)amino)-2-(2-

(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carbox-

ylate (0.21 g, 103%) as an orange foam which was used in the next

step without further puriﬁcation. LCMS (method high pH):

1: A mixture of (1R*,2R*,3S*,5S*)-tert-butyl 3-amino-2-(2-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]-

octane-8-carboxylate (16a) (0.22 g, 0.57 mmol), 2-(benzyloxy)-8-

chloro-3-methyl-1,7-naphthyridine (19a) (0.15 g, 0.53 mmol),

sodium tert-butoxide (0.152 g, 1.58 mmol), BrettPhos (0.028 g,

0.053 mmol), and Pd₂(dba)₃(0.024 g, 0.026 mmol) in THF (2 mL)

was stirred at room temperature for 1 h then at 60 °C for 18 h. The

mixture was cooled to room temperature and diluted with EtOAc (20

mL). 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: 0−80% EtOAc

in cyclohexane) gave (1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyl-

oxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-2-(2-(1,1-dioxidotetra-

hydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxy-

late (135 mg, 40%) as a pale-yellow foam. LCMS (method TFA):

1

Retention time 1.71 min, [M + H]⁺= 667.5 (1 Br). H NMR (400

MHz, CDCl₃) δ 7.98 (s, 1H), 7.96 (d, J = 1.2 Hz, 1H), 7.50-.46 (m,

2H) 7.45−7.39 (m, 2H), 7.38−7.32 (m, 1H), 5.92 (d, J = 9.0 Hz,

1H), 5.49 (s, 2H), 4.30 (br s, 3H), 3.87 (dd, J = 11.0, 2.7 Hz, 2H),

3.34−3.21 (m, 2H), 2.45 (d, J = 1.0 Hz, 3H), 2.15−1.99 (m, 2H),

1.99−1.79 (m, 3H), 1.78−1.30 (m, 8H), 1.55 (s, 9H), 1.29−1.08 (m,

3H). Step 3: A 20 mL microwave vial was charged with (5-

methylpyridin-3-yl)boronic acid (0.062 g, 0.45 mmol),

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-5-bromo-3-methyl-

1,7-naphthyridin-8-yl)amino)-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-

8-azabicyclo[3.2.1]octane-8-carboxylate (0.20 g, 0.30 mmol), K₂CO₃

(0.125 g, 0.901 mmol), Pd(OAc)₂(6.75 mg, 0.030 mmol),

cataCXium A (10.8 mg, 0.0300 mmol), then was ﬁlled with 1,4-

dioxane (4 mL) and water (2 mL). The resulting mixture was stirred

and degassed for 20 min with nitrogen. The mixture was then stirred

at 100 °C for 30 min under microwave irradiations then was 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 an

hydrophobic frit and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (25 g column, gradient

0−100% EtOAc in cyclohexane) gave (1S*,2S*,3R*,5R*)-tert-butyl 3-

((2-(benzyloxy)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-

8-yl)amino)-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-8-azabicyclo-

[3.2.1]octane-8-carboxylate (145 mg, 71%) as a pale-yellow gum. This

product was dissolved in TFA (2 mL), and the resulting solution was

stirred at reﬂux for 3 h then was cooled to room temperature and

concentrated in vacuo. The residue was dissolved in MeOH and

loaded onto a 5 g SCX2 cartridge, which was washed with MeOH (10

mL) then eluted with a 2N NH₃solution in MeOH. The eluant was

evaporated in vacuo to give 3-methyl-5-(5-methylpyridin-3-yl)-8-

(((1S*,2R*,3R*,5R*)-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-8-

azabicyclo[3.2.1]octan-3-yl)amino)-1,7-naphthyridin-2(1H)-one (92

mg, 63%) as a pale-yellow foam. LCMS (method high pH):

1

Retention time 1.06 min, [M + H]⁺= 635. H NMR (600 MHz,

CDCl₃) δ 7.84 (d, J = 5.6 Hz, 1H), 7.66 (d, J = 1.0 Hz, 1H), 7.51−

7.46 (m, 2H), 7.45−7.38 (m, 2H), 7.35 (d, J = 7.3 Hz, 1H), 6.70 (d, J

= 5.9 Hz, 1H), 5.90 (d, J = 9.8 Hz, 1H), 5.54−5.42 (m, 2H), 4.46−

4.18 (m, 3H), 2.94−2.70 (m, 4H), 2.40 (d, J = 1.0 Hz, 3H), 2.06 (s,

6H), 1.98−1.80 (m, 6H), 1.79−1.57 (m, 2H), 1.54 (s, 9H), 1.27 (m, J

= 7.2 Hz, 2H). Step 2: A solution of (1S*,2S*,3R*,5R*)-tert-butyl 3-

((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-2-(2-(1,1-di-

oxidotetrahydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-

8-carboxylate (0.13 g, 0.21 mmol) in CH₂Cl₂(10 mL) was cooled

using an ice bath then was treated with NBS (0.036 g, 0.205 mmol) in

two portions, of 22 mg and 14 mg, with 2 min stirring interval

between additions. The mixture was then treated with a 10% w/w

sodium metabisulphite aqueous solution, and the layers were

separated. The organic phase was dried using a phase separator and

concentrated in vacuo to give (1S*,2S*,3R*,5R*)-tert-butyl 3-((2-

(benzyloxy)-5-bromo-3-methyl-1,7-naphthyridin-8-yl)amino)-2-(2-

(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]-

octane-8-carboxylate (0.15 g, 103%) as an orange foam which was

used in the next step without further puriﬁcation. LCMS (method

1

high pH): Retention time 1.62 min. [M + H]⁺= 715.5 (1 Br). H

NMR (400 MHz, CDCl₃) δ 8.01−7.94 (m, 1H), 7.50−7.40 (m, 2H),

7.36 (d, J = 7.1 Hz, 1H), 5.89 (d, J = 9.5 Hz, 1H), 5.49 (d, J = 5.1 Hz,

2H), 4.45−4.08 (m, 3H), 3.02−2.80 (m, 4H), 2.46 (d, J = 1.0 Hz,

3H), 2.13−1.80 (m, 8H), 1.79−1.61 (m, 5H), 1.57−1.52 (m, 10H),

1.45 (s, 5H). Step 3: A 20 mL microwave vial was charged with (5-

methylpyridin-3-yl)boronic acid (0.043 g, 0.32 mmol),

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-5-bromo-3-methyl-

1,7-naphthyridin-8-yl)amino)-2-(2-(1,1-dioxidotetrahydro-2H-thio-

pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.15 g,

0.21 mmol), K₂CO₃(0.087 g, 0.63 mmol), Pd(OAc)₂(4.7 mg,

0.021 mmol), and di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine

(cataCXium A) (7.5 mg, 0.021 mmol) then was ﬁlled with 1,4-

dioxane (4 mL), and water (2 mL). The resulting mixture was stirred

and degassed for 20 min with nitrogen then was stirred at 100 °C for

30 min under microwave irradiations 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: 0−100%

EtOAc in cyclohexane) gave (1S*,2S*,3R*,5R*)-tert-butyl 3-((2-

(benzyloxy)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-8-

yl)amino)-2-(2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-8-

azabicyclo[3.2.1]octane-8-carboxylate (0.122 g, 80%) as a yellow

gum. LCMS (method high pH): Retention time 1.46 min, [M + H]⁺

1

Retention time 0.82 min, [M + H]⁺= 488.6. H NMR (400 MHz,

DMSO-d₆) δ 8.44 (d, J = 1.5 Hz, 1H), 8.37 (d, J = 1.8 Hz, 1H), 7.70

(s, 1H), 7.59−7.56 (m, 1H), 7.48 (d, J = 1.3 Hz, 1H), 6.55 (d, J = 8.1

Hz, 1H), 4.27 (br s, 1H), 3.80−3.71 (m, 2H), 3.64−3.57 (m, 1H),

3.53 (d, J = 3.8 Hz, 1H), 3.28−3.19 (m, 2H), 2.85 (br s 2H), 2.40 (s,

3H), 2.11 (d, J = 1.3 Hz, 3H), 2.04−1.96 (m, 1H), 1.91−1.65 (m,

6H), 1.64−1.44 (m, 4H), 1.44−1.28 (m, 2H), 1.28−1.17 (m, 1H),

1.16−1.00 (m, 2H). Step 4: The two enantiomers of 3-methyl-5-(5-

methylpyridin-3-yl)-8-(((1S*,2R*,3R*,5R*)-2-(2-(tetrahydro-2H-

pyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-1,7-naphthyr-

idin-2(1H)-one were separated by chiral chromatography. Analytical

method: approximately 0.5 mg of racemate were dissolved in 1:1

EtOH/heptane (1 mL). Then 20 μL were injected on column. Eluant:

50% EtOH (+0.2% isopropylamine) in heptane, f = 1.0 mL/min,

wavelength 215 nm. Column 4.6 mm i.d. × 25 cm Chiralpak IC.

Preparative method: 35 mg of racemate were dissolved in EtOH (1

mL) then were injected onto the column. Eluant: 40% EtOH (+0.2%

isopropylamine) in heptane, f = 15 mL/min, wavelength = 215 nm.

Column 2 cm × 25 cm Chiralpak IC. Total number of injection: 1.

The fractions were collected to give 3-methyl-5-(5-methylpyridin-3-

yl)-8-(((1S,2R,3R,5R)-2-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-8-

azabicyclo[3.2.1]octan-3-yl)amino)-1,7-naphthyridin-2(1H)-one (22)

(13 mg, 74%) as the fastest running enantiomer and 3-methyl-5-(5-

methylpyridin-3-yl)-8-(((1R,2S,3S,5S)-2-(2-(tetrahydro-2H-pyran-4-

yl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-1,7-naphthyridin-

2(1H)-one (11 mg, 63%) as slowest running enantiomer.

1

= 726. H NMR (400 MHz, CDCl₃) δ 8.51−8.47 (m, 1H), 7.80 (s,

1H), 7.74−7.68 (m, 1H), 7.56−7. 47 (m, 3H), 7.46−7.40 (m, 3H),

7.36 (d, J = 7.3 Hz, 1H), 6.02 (d, J = 9.8 Hz, 1H), 5.56−5.46 (m,

2H), 4.52−4.38 (m, 2H), 4.37−4.12 (m, 2H), 2.99−2.74 (m, 6H),

2.44 (s, 3H), 2.37 (d, J = 1.0 Hz, 3H), 2.15−1.82 (m, 8H), 1.80−1.60

(m, 5H), 1.56 (s, 6H), 1.49−1.15 (m, 3H). Step 4: A solution of

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-3-methyl-5-(5-meth-

ylpyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-2-(2-(1,1-dioxidotetra-

hydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxy-

8-(((1S,2R,3R,5R)-2-(2-(1,1-Dioxidotetrahydro-2H-thiopyr-

an-4-yl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-3-methyl-

5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one (24). Step

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late (0.12 g, 0.16 mmol) in TFA (1 mL) was reﬂuxed for 4 h then was

allowed to stand at room temperature for 48 h before being

concentrated in vacuo. The brown residue obtained was puriﬁed by

MDAP (method high pH). The residue obtained was further puriﬁed

by MDAP (method formic) to give 8-(((1S*,2R*,3R*,5R*)-2-(2-

(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]-

octan-3-yl)amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyri-

din-2(1H)-one (60 mg, 68%) as a pale-yellow solid. LCMS (method

high pH): Retention time 0.74 min, [M + H]⁺= 536. ¹H NMR (400

MHz, CDCl₃) δ 8.52 (br s, 1H), 8.46 (br s, 1H), 7.85−7.79 (m, 1H),

7.78 (s, 1H), 7.59 (d, J = 3.7 Hz, 2H), 4.61−4.45 (m, 1H), 4.18 (d, J

= 4.9 Hz, 1H), 4.11 (br s, 1H), 3.12−2.88 (m, 4H), 2.48 (s, 3H), 2.33

(d, J = 12.0 Hz, 3H), 2.19 (s, 6H), 2.04 (d, J = 11.0 Hz, 3H), 1.78 (br

s, 4H), 1.40 (br s, 2H), 1.34−1.19 (m, 1H). Step 5: The two

enantiomers of 8-(((1S*,2R*,3R*,5R*)-2-(2-(1,1-dioxidotetrahydro-

2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-3-

methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one were

separated by chiral chromatography. Analytical method: Approx-

imately 0.5 mg of material were dissolved in 50% EtOH in heptane (1

mL) and 20 μL were injected onto the column. Eluant: 70% EtOH

(+0.2% isopropylamine) in heptane, f = 1.0 mL/min, wavelength =

215 nm. Column 4.6 mm i.d. × 25 m Chiralpak IC. Preparative

method: Approximately 50 mg of material were dissolved in EtOH (1

mL) and isopropylamine (100 μL). The solution was injected onto

the column. Eluant: 70% EtOH (+0.2% isopropylamine) in heptane, f

= 25 mL/min, wavelength 215 nm. Column 30 mm × 25 cm

Chiralpak IC. The appropriate fractions were collected and

concentrated in vacuo to give 8-(((1S,2R,3R,5R)-2-(2-(1,1-dioxidote-

trahydro-2H-thiopyran-4-yl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)-

amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-

one (24) (13 mg, 52%) as the fastest running enantiomer and 8-

(((1R,2S,3S,5S)-2-(2-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-

ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-3-methyl-5-(5-methyl-

pyridin-3-yl)-1,7-naphthyridin-2(1H)-one (13 mg, 52%) as the

slowest running enantiomer. Each product was obtained as pale-

yellow solid.

8-(((1S,2S,3S,5R)-2-(2-(4,4-Diﬂuorocyclohexyl)ethyl)-8-

azabicyclo[3.2.1]octan-3-yl)amino)-3-methyl-5-(5-methylpyri-

din-3-yl)-1,7-naphthyridin-2(1H)-one (26). Step 1: A mixture of

(1R*,2R*,3S*,5S*)-tert-butyl 3-amino-2-(2-(4,4-diﬂuorocyclohexyl)-

ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (16b), (0.36 g, 0.97

mmol), 2-(benzyloxy)-8-chloro-3-methyl-1,7-naphthyridine (19a)

(0.25 g, 0.88 mmol), sodium tert-butoxide (0.253 g, 2.63 mmol),

BrettPhos (0.094 g, 0.18 mmol), and Pd₂(dba)₃(0.080 g, 0.088

mmol) in THF (2 mL) was stirred at room temperature for 1 h then

at 60 °C for 18 h. The mixture was then cooled to room temperature

and diluted with EtOAc (20 mL). The mixture 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: 0−80% EtOAc in cyclohexane) gave (1S*,2S*,3R*,5R*)-

tert-butyl 3-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-yl)amino)-

2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octane-8-car-

boxylate (0.51 g, 94%). LCMS (method high pH): Retention time

1.27 min, [M + H]⁺= 621. ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J

= 5.6 Hz, 1H), 7.65 (d, J = 1.0 Hz, 1H), 7.52−7.46 (m, 2H), 7.46−

7.39 (m, 2H), 7.35 (d, J = 7.3 Hz, 1H), 6.70 (d, J = 5.6 Hz, 1H), 5.92

(d, J = 9.3 Hz, 1H), 5.49 (d, J = 2.9 Hz, 2H), 4.45−4.12 (m, 3H),

2.40 (d, J = 1.0 Hz, 3H), 2.20−1.83 (m, 8H), 1.80−1.58 (m, 7H),

1.55 (s, 9H), 1.42−1.06 (m, 5H). Step 2: A solution of

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-3-methyl-1,7-naph-

thyridin-8-yl)amino)-2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-

azabicyclo[3.2.1]octane-8-carboxylate (0.30 g, 0.48 mmol) in CH₂Cl₂

(10 mL) was cooled using an ice bath then was treated with the

portionwise addition of NBS (0.086 g, 0.48 mmol) with a 10 min

stirring interval between additions. The resulting mixture was stirred

for 20 min then was treated with a 10% w/w sodium metabisulphite

aqueous solution. The layers were separated, and the organic phase

was dried over MgSO₄and concentrated in vacuo to give

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-5-bromo-3-methyl-

1,7-naphthyridin-8-yl)amino)-2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-

azabicyclo[3.2.1]octane-8-carboxylate (0.35 g, 104%) as a pale-yellow

foam which was used in the next step without further puriﬁcation.

LCMS (method high pH): Retention time 1.84 min, [M + H]⁺=

1

699/701 (1 Br). H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H), 7.96

(d, J = 1.0 Hz, 1H), 7.50−7.46 (m, 2H), 7.45−7.39 (m, 2H), 7.35 (d,

J = 7.1 Hz, 1H), 5.91 (d, J = 9.5 Hz, 1H), 5.49 (d, J = 2.4 Hz, 2H),

4.31 (m, 3H), 2.45 (d, J = 1.0 Hz, 3H), 2.13−1.79 (m, 8H), 1.75−

1.46 (m, 14H), 1.43- 1.06 (m, 7H). Step 3: A 20 mL microwave vial

was charged with (5-methylpyridin-3-yl)boronic acid (0.103 g, 0.750

mmol), (1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-5-bromo-3-

methyl-1,7-naphthyridin-8-yl)amino)-2-(2-(4,4-diﬂuorocyclohexyl)-

ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.35 g, 0.50 mmol),

K₂CO₃(0.207 g, 1.50 mmol), Pd(OAc)₂(0.011 g, 0.050 mmol), and

di((3S,5S,7S)-adamantan-1-yl)(butyl)phosphine (cataCXium A)

(0.018 g, 0.050 mmol) then was ﬁlled with 1,4-dioxane (4 mL),

and water (2 mL). The resulting mixture was stirred and degassed for

20 min with nitrogen, was stirred at 100 °C for 30 min under

microwave irradiations, and then was 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: 0−100% EtOAc in cyclohexane) gave

(1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-3-methyl-5-(5-meth-

ylpyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-2-(2-(4,4-

diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

(0.33 g, 93%) as a yellow gum. LCMS (method high pH): Retention

time 1.68 min, [M + H]⁺= 712. ¹H NMR (400 MHz, CDCl₃) δ 8.49

(d, J = 2.0 Hz, 1H), 8.48 (d, J = 1.5 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J =

1.0 Hz, 1H), 7.54 (s, 1H), 7.51−7.47 (m, 2H), 7.45−7.40 (m, 2H),

7.37−7.31 (m, 1H), 6.05 (d, J = 9.5 Hz, 1H), 5.51 (d, J = 2.4 Hz,

2H), 4.30 (br s, 3H), 2.44 (s, 3H), 2.37 (d, J = 0.7 Hz, 3H), 2.04−

1.83 (m, 7H), 1.76−1.59 (m, 6H), 1.56 (s, 10H), 1.33−1.06 (m, 6H).

Step 4: A solution of (1S*,2S*,3R*,5R*)-tert-butyl 3-((2-(benzyloxy)-

3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-8-yl)amino)-2-

(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octane-8-carbox-

ylate (0.33 g, 0.46 mmol) in TFA (3 mL) was reﬂuxed for 5 h then

was cooled to room temperature and concentrated in vacuo. The

residue was loaded onto a 10 g SCX2 cartridge, which was washed

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

(20 mL). The eluant was concentrated in vacuo to give 8-

(((1S*,2R*,3R*,5R*)-2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-

azabicyclo[3.2.1]octan-3-yl)amino)-3-methyl-5-(5-methylpyridin-3-

yl)-1,7-naphthyridin-2(1H)-one (0.18 g, 74%) as a white foam.

LCMS (method high pH): Retention time 1.01 min, [M + H]⁺= 522.

¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J = 1.5 Hz, 1H), 8.37 (d, J

= 2.0 Hz, 1H),7.69 (s, 1H), 7.61 (s, 1H), 7.51 (d, J = 1.2 Hz, 1H),

6.72 (d, J = 8.1 Hz, 1H), 4.22−4.07 (m, 1H), 3.49−3.18 (m, 5H),

2.38 (s, 3H), 2.08 (d, J = 1.0 Hz, 3H), 1.98−1.84 (m, 3H), 1.81−1.46

(m, 10H), 1.38−1.12 (m, 4H), 1.03−0.94 (m, 2H). Step 5: The two

enantiomers o f 8-(((1 S*, 2R*, 3R *, 5R *)-2-(2-(4, 4-

diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-3-

methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one were

separated by chiral chromatography. Analytical method:: Approx-

imately 0.5 mg of racemate were dissolved in 50% EtOH in heptane

(1 mL) and 20 μLwere injected onto the column. Eluant: 10% EtOH

(+0.2% isopropylamine) in heptane, f = 1.0 mL/min, wavelength =

215 nm. Column 4.6 mm i.d. × 25 cm Chiralcel OD-H. Preparative

method: Approximately 160 mg were dissolved in EtOH (3 mL) and

isopropylamine (1 mL). Then 1 mL of the solution was injected onto

the column. Eluant: 10% EtOH (+0.2% isopropylamine) in heptane, f

= 30 mL/min, wavelength 215 nm. Column 30 mm × 25 cm

Chiralcel OD-H. Total number of injections: 5. The fractions were

collected and concentrated in vacuo to give 8-(((1R,2R,3R,5S)-2-(2-

(4,4-diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-

3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-one (76

mg, 95%) as the fastest running enantiomer and 8-(((1S,2S,3S,5R)-

2-(2-(4,4-diﬂuorocyclohexyl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)-

amino)-3-methyl-5-(5-methylpyridin-3-yl)-1,7-naphthyridin-2(1H)-

one (25) (71 mg, 89%) as the slowest running enantiomer.

M

DOI: 10.1021/acs.jmedchem.8b00862

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8-(((3S,4r,5R)-3,5-Dimethoxypiperidin-4-yl)amino)-3-meth-

yl-1,7-naphthyridin-2(1H)-one (30). Step 1: A mixture of

(3R,4r,5S)-tert-butyl 4-amino-3,5-dimethoxypiperidine-1-carboxylate

(18a) (0.20 g, 0.77 mmol), 2-(benzyloxy)-8-chloro-3-methyl-1,7-

naphthyridine (19a) (0.14 g, 0.49 mmol), sodium tert-butoxide

(0.142 g, 1.48 mmol), BrettPhos (0.026 g, 0.049 mmol), and

Pd₂(dba)₃(0.023 g, 0.025 mmol) in THF (5 mL) was stirred at room

temperature for 1 h, then at 60 °C for 2 h before being cooled to

room temperature and diluted with EtOAc (20 mL). The organic

phase was washed with water, dried over MgSO₄, and concentrated in

vacuo to give (3S,4r,5R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-

naphthyridin-8-yl)amino)-3,5-dimethoxypiperidine-1-carboxylate, a

brown residue (100 mg, 40%) as a brown oil which was used in

the next step without puriﬁcation. LCMS (method high pH):

Retention time 1.41 min, [M + H]⁺= 509. Step 2: A solution of

(3S,4r,5R)-tert-butyl 4-((2-(benzyloxy)-3-methyl-1,7-naphthyridin-8-

yl)amino)-3,5-dimethoxypiperidine-1-carboxylate (80 mg, 0.16

mmol) in TFA (3 mL) was reﬂuxed for 3 h then was cooled to

room temperature and concentrated in vacuo. The residue was

dissolved in MeOH (3 mL) and loaded onto a 5 g SCX cartridge.

This was washed with MeOH (20 mL) and then eluted with a 2N

NH₃solution in MeOH (20 mL). The eluant was concentrated in

vacuo to give 8-(((3S,4r,5R)-3,5-dimethoxypiperidin-4-yl)amino)-3-

methyl-1,7-naphthyridin-2(1H)-one (27) (48 mg, 96%) as a beige

solid. LCMS (method high pH): Retention time 0.60 min, [M + H]⁺

= 319. ¹H NMR (400 MHz, CDCl₃) δ 13.3−13.0 (br s, 1H), 8.01 (d,

J = 5.4 Hz, 1H), 7.67 (d, J = 1.0 Hz, 1H), 6.72 (d, J = 5.4 Hz, 1H),

6.45−6.28 (m, 1H), 4.59−4.43 (m, 1H), 3.48−3.35 (m, 11H), 2.74−

2.61 (m, 2H), 2.39 (d, J = 1.0 Hz, 3H).

Present Address

^⊥For R.J.S.: Oncology Innovative Medicines and Early

Development, 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 to Dr. Richard Upton for NMR support and Phylicia

Dassardo Joseph and Abigail Lucas for crystallization support.

ABBREVIATIONS USED

■

ANCCA, AAA nuclear coregulator cancer-associated protein;

ATAD2, ATPase family AAA domain containing 2; BET,

bromodomain and extra terminal domain; BRD4/7/9,

bromodomain-containing protein 4/7/9; BRPF1, bromodo-

main and PHD ﬁnger containing 1; CBP, CREB binding

protein; CREB, cAMP response element-binding protein;

EP300, histone acetyltransferase p300; KAc, acetyl lysine;

pIC₅₀, −log₁₀IC₅₀; SMARCA2/4, SWI/SNF related, matrix

associated, actin dependent regulator of chromatin, subfamily

a, member 2/4

REFERENCES

■

ASSOCIATED CONTENT

■

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Cosset, F.-L.; Verhoeyen, E.; Emadali, A.; Schweifer, N.; Gianni, D.;

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b00862.

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AUTHOR INFORMATION

■

Corresponding Authors

*For P.B.: phone, +44 1438 763246; fax, +44 1438 763352; E-

mail, Paul.A.Bamborough@gsk.com.

*For E.H.D.: phone, +44 1438 764319; fax, +44 1438 768302;

E-mail, Emmanuel.H.Demont@gsk.com.

ORCID

Paul Bamborough: 0000-0001-9479-2894

N

DOI: 10.1021/acs.jmedchem.8b00862

J. Med. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jmedchem.8b00862

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Robson, S.; Sheppard, R. J.; Upton, R.; Watson, R. J. A chemical

probe for the ATAD2 bromodomain. Angew. Chem., Int. Ed. 2016, 55,

11382−11386.

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http://www.thesgc.org/ (accessed June 28, 2018).

P

DOI: 10.1021/acs.jmedchem.8b00862

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Aiming to Miss a Moving Target: Bromo and Extra Terminal Domain (BET) Selectivity in Constrained ATAD2 Inhibitors

DOI: 10.1021/acs.jmedchem.8b00862

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.8b00862

Full text of DOI:10.1021/acs.jmedchem.8b00862

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