Identification and Characterization of von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1

Article abstract of DOI:10.1021/acs.jmedchem.7b00635

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC₅₀ = 12 nM, D_max = 96%) with excellent selectivity against a related kinase IKK?, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.

Full text of DOI:10.1021/acs.jmedchem.7b00635

Article
pubs.acs.org/jmc
Identification and Characterization of Von Hippel-Lindau-Recruiting
Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1
Andrew P. Crew,*,† Kanak Raina,† Hanqing Dong,† Yimin Qian,† Jing Wang,† Dominico Vigil,†,∥
Yevgeniy V. Serebrenik,‡ Brian D. Hamman,† Alicia Morgan,† Caterina Ferraro,† Kam Siu,†
Taavi K. Neklesa,† James D. Winkler,† Kevin G. Coleman,† and Craig M. Crews*,‡,§
†Arvinas LLC, 5 Science Park, New Haven, Connecticut 06511, United States
§
‡Department of Molecular, Cell and Developmental Biology, Departments of Chemistry and Pharmacology, Yale University, New
Haven, Connecticut 06511, United States
S
* Supporting Information
ABSTRACT: Proteolysis targeting chimeras (PROTACs) are
bifunctional molecules that recruit an E3 ligase to a target
protein to facilitate ubiquitination and subsequent degradation
of that protein. While the field of targeted degraders is still
relatively young, the potential for this modality to become a
differentiated and therapeutic reality is strong, such that both
academic and pharmaceutical institutions are now entering this
interesting area of research. In this article, we describe a
broadly applicable process for identifying degrader hits based
on the serine/threonine kinase TANK-binding kinase 1
(TBK1) and have generalized the key structural elements
associated with degradation activities. Compound 3i is a
potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent
selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras
cancer cells.
Proteolysis targeting chimeras (PROTACs),1−9 are a class of
INTRODUCTION
■
bifunctional molecules that exist outside of the “rule of 5”
The most common therapeutic interventions available to the
prescribing physician are inhibitor-based drugs such that the
active pharmaceutical ingredient mediates the function of the
aberrant protein via direct or allosteric inhibition of the
mechanistic activity of the said protein. Although inhibition
of protein activity is a clinically validated approach, there are
(Ro5)10 space that hijack the endogenous protein homeostasis
machinery. As illustrated in Figure 1, PROTACs can recruit an
E3 ubiquitin ligase to a target protein of interest (PoI),
resulting in polyubiquitination of the PoI, which is subsequently
degraded via the proteasome.11 We have recently demonstrated
the mechanism and the efficacy of our PROTAC technology by
significant constraints to its wider applicability. First, it usually
carries the burden of requiring protracted target engagement
for the mechanism and consequential function to be effectively
abrogated. Many protein−small molecule interactions are
associated with rapid off-rates, resulting in very low inhibitor
occupancy of the protein active site and inadequate down-
regulation of downstream signaling. Second, an inability to
reach tolerated free-drug concentrations at or above the in vitro
IC90, because of high plasma protein binding, poor
pharmacokinetics, or toxicity, can limit the effectiveness of
inhibitor drugs. Finally, many proteins possess little or no
mechanistic activity, yet execute their biological role by
providing a scaffolding function, and as a result, these proteins
are less susceptible to the inhibitor paradigm. For the reasons
listed above, technologies that can reduce levels of a target
protein in a manner that requires only transient interactions
with the protein could provide significant therapeutic utility.
targeting a variety of proteins of interest for degradation, in
both cultured cells and in vivo. These targets include the BET
(bromodomain and extra terminal domain) family protein
BRD4,12 the serine/threonine kinase RIPK2,13 and the
estrogen related receptor α (ERRα).13 In this article, we
describe for the first time the structural features governing the
potency of PROTAC molecules, especially the effect of varying
the effective distance between the two proteins by altering the
length of the connecting linker and modulating the binding
affinities to either the co-opted E3 ligase or target protein.
TANK-binding kinase 1 (TBK1) is a serine/threonine kinase
and a noncanonical member of the IKK family implicated in
Special Issue: Inducing Protein Degradation as a Therapeutic
Strategy
Received: May 4, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Protein degradation mediated by proteolysis targeting chimeras (PROTACs).
Figure 2. Structures of TBK1 ligands 1a and 1b, VHL ligand 2, and TBK1 PROTACs 3a−p.
diverse cellular functions, including innate immune response as
well as tumorigenesis and development, that has attracted
considerable attention with regard to the identification of
agents that could diminish its activity.14 Specifically, interest
converged upon this protein in the wake of the purported
critical role that TBK1 signaling plays in K-Ras mutant tumors
as elucidated using RNAi.15 However, subsequent reports
challenged this hypothesis, and the synthetic lethality of TBK1
knockdown in this context was called into question.16 In this
study, we embarked on a campaign to assess whether TBK1
could be degraded using PROTAC technology to delineate the
important structural features of active TBK1 PROTACs, with
emphasis on each of the three components (target ligand, E3
ligase ligand, and linker), and finally, to determine whether a
TBK1 PROTAC could replicate the K-Ras synthetic lethality
reported with TBK1 RNAi.
TBK1 structure, in concert with the available SAR, indicated
that an alternate egress point was available for linker
attachment from the ligand out to solvent from the para-
position of the pyrimidine-2-aminophenyl, and that a simple
alkyl ether chemistry should be tolerated at this position.
Additionally, it appeared that the NH of the secondary amide of
compound 1a was not providing any productive interactions
with TBK1 and was likely adding a desolvation burden to the
binding event suggesting an N-methyl derivative might be a
beneficial modification. Combining these changes along with a
bromo group in the 5-position of pyrimidine provided
compound 1b as a starting TBK1 PTM, which was confirmed
to be a potent binder to TBK1 with a Kd of 1.3 nM.
One of the E3 ligases with exciting therapeutic potential is
the von Hippel-Lindau (VHL) tumor suppressor, which exists
as part of an active E3 ubiquitin ligase complex.18 Compound 2
is an example of a hydroxyproline derivative that binds to VHL
to disrupt the VHL−HIF1α interaction19 with an IC50 of 0.8
μM in a fluorescence polarization (FP) assay. Structural
information on a related analogue bound to the VHL-
elongin/B-elongin C (VBC) complex (PDB code: 4W9H)20
suggested the acetamide group in 2 would be tolerant to
substitution and a good egress position from the protein for a
linker. With both ligands 1b and 2 in hand and the linker
RESULTS AND DISCUSSION
■
PROTAC molecular architecture requires a protein targeting
moiety (PTM) connected via a linker to an E3 ligase ligand.
For the TBK1 PTM, we selected a classic kinase amino-
pyrimidine chemotype for which a crystal structure of TBK1
bound to Compound 1a was available (PDB code 4IM0).17
Interrogation of the binding mode of compound 1a in the
B
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 1. Effect of Linker Length on the Degradation Activity of TBK1 PROTACs
a
NA: not applicable. ND: not determined. DC50: concentration at which 50% degradation is observed. Dmax: maximal degradation observed. Data
represent the mean of ≥2 determinations. PSA calculations performed with ChemAxon software.
connection positions determined, we carried out a PROTAC
design and synthesis campaign (3a−p, Figure 2).
required to allow the proteins to come together without
incurring steric conflicts. We hypothesize that the very flexible
nature of the linker chemistry allows even the very long linkers
to orient themselves so that the two proteins become associated
for ubiquitin to transfer to TBK1. In addition, because each
linker has a different composition and length, there will
necessarily be differing contacts with the protein−protein
interface which in turn will influence how strongly each
derivative PROTAC can bind to and stabilize the ternary
complex. This likely governs the efficiency of the key ubiquitin
transfer and therefore contributes to the subtle degradation
SAR seen across PROTACs 3f−3p, along with differences in
physicochemical properties. The formation of such a ternary
complex mediated through bifunctional PROTACs was
confirmed in the recently published crystal structure of MZ1,
a BET PROTAC with a JQ1 carboxamide linked to VHL ligand
2,21 bound to both VHL and BRD4 proteins.22
We employed flexible and therefore accommodating alkyl
ether chemistries to connect ligands 1b and 2, and not knowing
a priori at what distance these ligands would have to be
positioned in the PROTAC to effectively associate their
respective proteins, we undertook a systematic survey of
connector length. Table 1 lists the measured degradation
potency (DC50), maximum degradation observed (Dmax), and
the calculated two-dimensional polar surface area (PSA). From
this initial library set, several PROTACs (3f−3p) were
identified that degrade TBK1 with submicromolar potency.
The gross SAR clearly indicates a dependence on a minimum
linker length as PROTACs with linkers of <12 atoms
demonstrated no appreciable degradation activity (3a−3e).
Longer linkers appear generally well tolerated despite their
higher PSA and presumed cell penetrance burden, such that
even the 29-atom linker PROTAC 3p still provided robust
degradation albeit with reduced potency. These observations
are consistent with the concept that the bifunctional PROTAC
species mediates the association of the TBK1 and VHL proteins
to form a ternary complex but that a minimum linker length is
To confirm the mechanistic dependence on VHL for TBK1
degradation, we prepared PROTAC 4 (Figure 3), an epimer of
active PROTAC 3i, which by nature of the reversed (S)
stereochemistry at the proline 4-position has no appreciable
C
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Structures of TBK1 degrader 3i and its epimer 4.
binding to VHL (FP IC50 > 100 μM) but retained comparable
TBK1 binding (Kd = 5.9 nM for 4 versus 4.6 nM for 3i).
PROTAC 4 showed no significant degradation of TBK1
(Figure 4), confirming VHL’s role in the degradation of TBK1
both PROTAC agents as well as the parent TBK1 ligand 1b
confirmed competent intracellular TBK1 binding as indicated
by the inhibition of pIRF3. The significantly more potent
suppression of IRF3 phosphorylation by 3i compared to 4 is
indicative of the additional degradation activity afforded by 3i,
while 4 only exerted an antagonist effect.
The involvement of the proteasome in the VHL-mediated
degradation of TBK1 by PROTAC 3i was assessed by the
addition of the proteasome inhibitor carfilzomib to the assay
conditions. Pretreatment (30 min) with carfilzomib markedly
reduced the extent of TBK1 degradation by PROTAC 3i
indicating that the 26S proteasome was indeed implicated in
the degradation of TBK1 (Figure 5). Furthermore, addition of
excess VHL ligand 2 to the assay to compete with PROTAC 3i
for VHL binding also abrogated TBK1’s degradation.
With mechanistically specific tool degrader 3i in hand, we
next evaluated the impact of TBK1 binding affinity on
degradation potency and efficiency. In order to minimize the
impact of any cell permeation or conformational differences on
observed degradation potency, we modified only the 5-position
of the pyrimidine TBK1 ligand component and only used
functionalities that did not substantially alter the polar surface
Figure 4. PROTAC 3i but not its VHL-incompetent epimer 4 nor
TBK1 inhibitor 1b effects the degradation of TBK1. All three display
competent intracellular TBK1/pIRF3 activity.
by PROTAC 3i. The effect of 3i and 4 on the TBK1
downstream marker pIRF3 was also assessed. Stimulation of
Panc02.13 cells with Toll-like receptor 3 (TLR3) agonist
polyinosine-polycytidylic acid [poly(I:C)] in the presence of
Figure 5. PROTAC 3i mediated degradation of TBK1 is abrogated by pretreatment with either VHL ligand 2 or proteasome inhibitor carfilzomib
(Carf).
D
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 2. Effect of TBK1 Affinity on Degradation Activity
compd
R1
TBK1 Kd (nM)
DC50 (nM)
Dmax (%)
5a
5b
5c
5d
5e
5f
H
725
103.5
10.4
4
>1000
282
10
ND
74
96
96
96
89
77
70
96
96
96
F
Cl
I
3
CF3
Me
Et
13
29
270
275
1035
130
245
4.6
92
5g
5h
5i
121
544
48
cBu
vinyl
cPr
Br
5j
65
3i
12
a
ND: not determined. DC50: concentration at which 50% degradation is observed. Dmax: maximal degradation observed. Data represent the mean of
≥2 determinations.
Table 3. Effect of VHL Affinity on Degradation Activity
cmpd
R2
PROTAC FP VHL IC50 (μM)
DC50 (nM)
Dmax (%)
6a
6b
6c
6d
6e
3i
H
>100
25.0
9.9
>1000
>1000
864
288
44
0
Me
34
71
75
88
96
Et
nPr
iPr
tBu
16.0
5.5
4.9
9
area of the set. Table 2 lists TBK1 binding affinities and the
TBK1 degradation activity of these PROTACs.
in the clinic where attaining a sufficiently high serum
concentration of an inhibitor is a critical component for
efficacy. Coupled with the longer-lasting effects of protein
degradation on downstream signaling that we have already
demonstrated elsewhere,12,13 this result considerably strength-
ens the case for applying our technology to targets that prove
intractable using conventional approaches and/or targets with
low affinity ligands.
Next, we determined the effect of altering PROTAC VHL
affinity, as measured in a fluorescence polarization (FP) assay,
on PROTAC mediated degradation (Table 3). PROTACs 3i
and 6a−6e differ in the side chain chemistry of the glycine
component of the VHL ligand which, as for the TBK1 ligand,
do not grossly change the molecular properties of the
Maximal efficacy (>90% degradation) was achieved with
PROTACs that had TBK1 affinities (Kd) less than 245 nM,
beyond which degradation begins to drop off, although
remaining significant (70%) even in the case of compound
5h that has a Kd of 1 μM. The surprisingly high cellular
degradation potency (DC50 = 65 nM) seen with 5j, in spite of
its modest affinity for TBK1 (Kd = 245 nM) and the relatively
high IC50 value of its VHL-binding component ligand (IC50
=
800 nM), is likely due in part to the ability of the PROTAC to
initiate multiple cycles of target protein degradation.12 This is a
mechanistic advantage of PROTAC technology over the
traditional inhibitor paradigm that might prove to be of utility
E
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
PROTACs (such as PSA) yet do alter their VHL affinity.
Maximal efficacy was only seen with the parent PROTAC 3i (R
= tert-butyl; VHL ligand IC50 = 4.9 μM), although robust
degradation (>70%) was also seen with PROTACs 6c and 6d
that displays up to 3-fold weaker VHL affinity. Interestingly, the
fact that PROTAC 6b did not show robust degradation activity
despite displaying VHL affinity similar to that of 6d is
consistent with the operation of the more complex ternary
system that that requires cooperative binding of the PROTAC
to both proteins in play and suggests that the methyl group in
6b is even more inferior at supporting this complex than the
already suboptimal ethyl and n-propyl groups in 6c and 6d,
respectively.
We next tested the ability of potent PROTAC 3i to degrade
the noncanonical IkB kinase IKKε, a close homologue of
TBK1, with which it shares 65% similarity. Since the TBK1
ligand used in the design of PROTAC 3i exhibits poor
selectivity for TBK1 over IKKε (IC50 of 1.3 nM vs 8.7 nM), we
expected substantial degradation of IKKε in our assays. To our
surprise, PROTAC 3i had no effect on the levels of IKKε, at
concentrations of more than 50-fold above its TBK1 DC50
(Figure 6). We verified that PROTAC 3i was capable of
therefore a different pattern and/or efficiency of the transfer of
ubiquitin to TBK1 (Figure 1). While such a difference in the
formation and stability of the active trimer complexes could
contribute to the selectivity, other potential explanations for
this observation include an increased rate of deubiquitinylation
in the case of IKKε or that IKKε ubiquitinylation by VHL
results in an outcome other than protein degradation.
Ubiquitination as a post-translational modification is known
to serve a large variety of cellular functions,24 and such an effect
in the case of IKKε would be of considerable interest. A
detailed investigation, however, is beyond the scope of the
current article and will be left to a subsequent publication.
We next evaluated the effect of potent TBK1 degrader 3i on
cell lines harboring either wild-type or mutant K-Ras.
Treatment of both K-Ras mutant cell lines (H23, A549, and
H1792) and K-Rras wild type cell lines (H2110 and HCC827)
with PROTAC 3i for 72 h, while affecting near complete
degradation of TBK1, caused no differential effect on the
proliferation of these cells (Figures 7 and 8). This result is
consistent with the literature reports that TBK1 was not
synthetically lethal in K-Ras mutant versus wild type cells.16
Chemistry. The synthesis of TBK ligand 1b is shown in
Scheme 1. Starting from commercially available N-BOC
protected amine 7, amidation with cyclobutanecarboxylic acid,
and subsequent removal of the BOC group generated amine 8,
which selectively displaced the 4-Cl of 5-bromo-2,4-dichlor-
opyrimidine to afford compound 9. Further SNAr reactions with
4-ethoxyaniline and 4-aminophenol under acidic conditions
yielded the TBK1 ligand 1b and key synthetic intermediate 10.
The VHL ligand 2 was prepared according to literature
procedures,14,15 and the preparation of VHL amino building
blocks 18a−f is shown in Scheme 2. Following Pd-catalyzed
arylation of 4-bromobenzonitrile (11) and subsequent
reduction with LiAlH4, benzyl amine 13 was formed, which
was coupled with N-BOC hydroxyproline (14) to give
compound 15. Upon deprotection of 15 with HCl/MeOH
and subsequent amide formation with an α-substituted N-
BOC-glycine, intermediates 17a−f were generated, which were
BOC-deprotected to afford the corresponding VHL amino
building blocks 18a−f.
Scheme 3 illustrates two general approaches to synthesize
PROTACs 3a−p. In method A, the tosyl group of intermediate
20 was displaced by 4-nitrophenol, and the nitro group was
reduced to provide amine 22. This amine was reacted with 2-
chloropyrimidine 9 to afford ester 23, which was converted to
its carboxylic acid and then coupled with VHL building block
18f to afford the desired PROTACs. In method B, compound
23 was obtained in one step through the direct displacement of
the tosyl group in 20 by the key intermediate 10.
The syntheses of epimeric PROTAC 4 and PROTAC
analogues 6a−e are described in Scheme 4. Following the
similar sequence as described in Scheme 2, the epimeric VHL
amine building block 27 was prepared. The acid intermediate
24i was coupled with different VHL amino building blocks 27
and 18a−e to afford PROTACs 4 and 6a−e, respectively.
For the exploration of the SAR of different TBK1 ligands, the
acid derivative of linker 20i was first coupled with the VHL
amino building block 18f, and the resulting tosylate 28 reacted
with different TBK1 phenols 29a−j to generate the
corresponding PROTACs 5a−j as displayed in Scheme 5.
Debromination of 10 was achieved by hydrogenation over Pd−
C to give phenol 29a. For PROTACs with F, Cl, I, CF3, Me,
and Et at the 5-position of the pyrimidine ring, commercially
Figure 6. PROTAC 3i selectively degrades TBK1 over IKKε.
binding IKKε (Kd = 70 nM) and that the observed band was
IKKε through knock down with siRNA (Figure 6). As
expected, scramble (scr) siRNA did not affect the IKKε levels,
while three siRNAs completely knocked out the protein (Figure
6).
Since the reported Km values for ATP are not strikingly
different for the two kinases,23 we hypothesized that this
introduction of degradation selectivity into a relatively
unselective ligand likely results from a differential presentation
of TBK1 and its surface lysines to VHL and its reactive E2-
ubiquitin thioester component, as compared to IKKε, and
F
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 7. TBK1 degradation in K-Ras mutant and wild type cells (16 h of treatment).
PROTACs can provide greater potency and selectivity than that
anticipated based on the potency and selectivity of the
component ligands. As targeted protein degradation with
PROTACs and other molecules utilizing a similar mechanism
has gained wider currency and validity in drug discovery, the
study detailed herein could be used as a template for identifying
degrader hits and leads, although for each protein targeted and
ligase recruited, the affinity and linker length requirements will
likely be different. Indeed, using this approach, we have
successfully identified degrader leads across a variety of target
protein classes, and our current efforts focused on optimizing
those leads into orally active drug candidates. In subsequent
publications, we will describe the breadth of this platform
technology as it relates to other targets of interest and the
achievement of oral activity.
Figure 8. Relative proliferation of K-Ras mutant and wild type lung
cancer cell lines in the presence of TBK1 degrader 3i (72 h treatment;
10 μM top concentration).
available 2,4-dichloropyrimidines 30 were deployed, and a
similar chemistry was followed to generate compounds 29b−g.
The introduction of cyclobutyl, vinyl, and cyclopropyl groups
was achieved through Suzuki coupling reactions with 9,
followed by SNAr displacement by 4-aminophenol to give
compounds 29h−j.
EXPERIMENTAL SECTION
■
General Chemical Methods. All reagents were obtained from
commercial suppliers and used without further purification. Flash
chromatography was performed using an ISCO CombiFlash RF 75
PSI with RediSep normal-phase silica gel cartridges. Preparative HPLC
purification was performed on a Waters UV-directed purification
system equipped with 2545 Binary Gradient Module. The mobile
phases were water (0.1%TFA or 0.01% NH4HCO3) and acetonitrile
CONCLUSIONS
■
1
with a flow rate of 30 mL/min. H NMR (300 or 400 MHz) and 13C
Herein, we have described a process for the rapid generation of
potent, VHL, and proteasome-dependent PROTACs as
degraders of TBK1, through a systematic survey of linker
length and ligand affinities. We have also demonstrated that
NMR (100.6 MHz) spectra were recorded on Bruker spectrometers.
Analytical LC-MS data were collected on a Shimadzu LCMS-2020
with mobile phases as acetonitrile and water containing 0.05% TFA.
a
Scheme 1. Syntheses of TBK1 Ligand 1b and Key Intermediate 10
a
Reagents and conditions: (a) cyclobutanecarboxylic acid, HATU, DIPEA, DMF, rt, overnight; (b) HCl, MeOH, rt, 1 h; (c) 5-bromo-2,4-
dichloropyrimidine, DIPEA, MeCN, rt, 3 h; (d) substituted anilines, dioxane, TsOH, 90 °C, overnight.
G
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Syntheses of VHL Amino Building Blocks
a
Reagents and conditions: (a) 4-methylthiazole, Pd(OAc)2, DMA, 150 °C, 5 h; (b) LiAlH4, THF, reflux, 5 h; (c) 14, HATU, DIPEA, DMF, rt, 2 h;
(d) HCl, MeOH, 2 h; (e) N-BOC amino acids, HATU, DIPEA, DMF, rt, 3 h; (f) HCl, MeOH, rt, 3 h.
a
Scheme 3. Syntheses of TBK1 PROTACs 3a−3p
a
Reagents and conditions: (a) p-TsCl, NEt3, cat. DMAP, DCM, rt, overnight; (b) 4-nitrophenol, K2CO3, DMF, 70 °C, overnight; (c) Pd/C, H2,
EtOH, rt, overnight; (d) 9, p-TsOH, dioxane, 100 °C, overnight; (e) 4 N HCl/dioxane, rt, overnight; (f) 18f, HATU, DIPEA, DMF, rt, 1 h; (g)
Cs2CO3, 80 °C, overnight.
The purities of all final compounds were over 95% as determined by
LC-MS analysis monitored at 214 and 254 nM.
1.87 (m, 3H), 1.34 (t, J = 7.0 Hz, 3H). LC-MS (ES+): m/z 461.9 and
463.9 [M + H+].
General Procedure for Preparing PROTACs Using Method A
As Described in Scheme 3. (2S,4R)-1-[(2S)-2-[2-(4-{4-[(5-Bromo-4-
{[3-(1-cyclobutyl-N-methylformamido)propyl]amino}pyrimidin-2-
yl)amino]phenoxy}butoxy)acetamido]-3,3-dimethylbutanoyl]-4-hy-
droxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-
carboxamide (3a). A mixture of 21a (600 mg, 1.8 mmol) and
palladium on carbon (10%, 60 mg) in ethanol (20 mL) was stirred
under hydrogen atmosphere at rt overnight. The mixture was filtered
and washed with ethanol. The filtrate was concentrated under reduced
pressure to afford tert-butyl 2-(4-(4-aminophenoxy)butoxy)acetate
(22a) as a colorless oil (550 mg, 90% yield). This oily material (82 mg,
0.28 mmol) was mixed with compound 9 (100 mg, 0.28 mmol) and
N-[3-({5-Bromo-2-[(4-ethoxyphenyl)amino]pyrimidin-4-yl}-
amino)propyl]-N-methylcyclobutanecarboxamide (1b). A solution
containing 9 (50 mg, 0.14 mmol), 4-ethoxyaniline (47 mg, 0.35
mmol), 4 N HCl in 1,4-dioxane (0.10 mL), and n-butanol (1 mL) was
heated in a microwave oven at 120 °C for 40 min. The reaction
mixture was concentrated, and the residue was purified using a
Teledyne Combiflash (0 to 3% 7 N NH3/MeOH in DCM) to afford
1
compound 1 as an off white solid (64 mg, 99% yield). H NMR (400
MHz, CD3OD) δ 7.83 (2s, 1H), 7.42 (d, J = 9.0 Hz, 2H), 6.78−6.87
(m, 2H), 3.97 (2q, J = 7.0 Hz, 2H), 3.31−3.47 (m, 4H), 3.17−3.26 (m,
1H), 2.84 (2s, 3H), 2.10−2.24 (m, 3H), 1.88−2.03 (m, 2H), 1.69−
H
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Syntheses of TBK1 PROTACs 4 and 6a−e
a
Reagents and conditions: (a) 13, HATU, DIPEA, DMF, rt, overnight; (b) HCl, MeOH, rt, 2 h; (c) N-Boc L-tert-leucine, HATU, DIPEA, DMF, rt,
overnight; (d) 27 or 18a−e, HATU, DIPEA, DMF, rt, 2 h.
a
Scheme 5. Syntheses of TBK1 PROTACs 5a−j
a
Reagents and conditions: (a) TFA, DCM, rt, 3 h; (b) 18f, HATU, DIPEA, DMF, rt, 2 h; (c) Pd/C, H2, MeOH, rt, 0.5 h; (d) 28, Cs2CO3, 80 °C, 5
h; (e) 8, DIPEA, MeCN, rt, overnight; (f) 4-aminophenol, p-TsOH, dioxane, 90 °C, overnight; (g) RB(OH)2, PdCl2(dppf), Na2CO3, toluene/water,
100 °C, 3 h.
toluene sulfonic acid monohydrate (19 mg, 0.1 mmol) in dioxane (3
mL), and the mixture was stirred at 100 °C for 16 h. The reaction
mixture was first cooled to rt and then partitioned between ethyl
acetate and aqueous NaHCO3 solution. The organic phase was washed
with water, brine, dried over anhydrous sodium sulfate, and
concentrated. The residue was purified by silica gel column
chromatography using 2%−5% MeOH in dichloromethane as eluent
t o a ff o r d t e r t - b u t y l 2 - ( 4 - ( 4 - ( 5 - b r o m o - 4 - ( 3 - ( N -
methylcyclobutanecarboxamido)propylamino)pyrimidin-2-ylamino)-
phenoxy)butoxy)acetate (23a) as a yellow oil (60 mg, 35% yield). LC-
MS (ES+): m/z 620.2 and 622.2 [M + H+]. This oil (60 mg, 0.1
mmol) in 4 M HCl/dioxane (2 mL) was stirred at rt for 1 h. The
reaction mixture was concentrated under reduced pressure to provide
2-(4-(4-(5-bromo-4-(3-(N-methylcyclobutanecarboxamido)-
propylamino)pyrimidin-2-ylamino)phenoxy)butoxy)acetic acid (24a)
as a yellow oil (55 mg, 95% yield). Carboxylic acid 24a (55 mg, 0.1
mmol) was mixed with 18f (45 mg, 0.1 mmol) and DIPEA (46 mg,
0.36 mmol) in dry DMF (3 mL). To this solution was added HATU
(74 mg, 0.2 mmol) at 0 °C. The resulting mixture was stirred at rt for
0.5 h. The mixture was partitioned between ethyl acetate and water.
I
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
The organic phase was washed with water and brine and dried over
anhydrous sodium sulfate, filtered, and concentrated. The residue was
purified by preparative TLC to afford the desired product 3a as a white
solid (19 mg, 20% yield). 1H NMR (400 MHz, CD3OD): δ 1.03, 1.05
(two singlets, 9H), 1.79−2.26 (m, 15H), 2.48 (s, 3H), 2.85−2.91 (2s,
3H), 3.27 (t, J = 7.2 Hz, 1H), 3.42−3.50 (m, 3H), 3.65 (t, J = 6.0 Hz,
2H), 3.79−4.07 (m, 6H), 4.34−4.39 (m, 1H), 4.52−4.62 (m, 3H),
4.72 (t, J = 6.8 Hz, 1H), 6.85−6.91 (m, 2H), 7.41−7.49 (m, 6H), 7.59
(d, J = 9.2 Hz, 1H, exchanged H), 7.87 (2s, 1H), 8.87 (s, 1H). LC-MS
(ES+): m/z 976.2/978.2 [M + H+].
7.87−7.93 (2s, 1H), 8.78(s, 1H). LC-MS (ES+): m/z 992.3 and 994.3
[M + H+].
(2S,4R)-1-[(2S)-2-{2-[3-(3-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenoxy}-
propoxy)propoxy]acetamido}-3,3-dimethylbutanoyl]-4-hydroxy-N-
{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
mide (3d). 1H NMR (400 MHz, CD3OD): δ 1.05 (t, J = 7.2 Hz, 9H),
1.38−1.41 (m, 4H), 1.83−1.92 (m, 3H), 1.98−2.12 (m, 4H), 2.18−
2.29 (m, 4H), 2.48 (2s, 3H), 2.88 (multiple s, 3H), 3.24 (m, 1H),
3.38−3.65 (m, 9H), 3.75−4.06 (m, 6H), 4.35 (d, J = 15.6 Hz, 1H),
4.49−4.62 (m, 3H), 4.71 (t, J = 6.8 Hz, 1H), 6.85−6.90 (m, 2H),
7.40−7.56 (m, 6H), 7.87 (2s, 1H), 8.87−8.90 (s and m, 1H). LC-MS
(ES+): m/z 1020.2 and 1022.2 [M + H+].
General Procedure for Preparing PROTACs Using Method B
As Described in Scheme 3. (2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-
{[3-(1-cyclobutyl-N-methylformamido)propyl]amino}pyrimidin-2-
yl)amino]phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-di-
methylbutanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)-
phenyl]methyl}pyrrolidine-2-carboxamide (3i). A mixture of tert-
butyl 2-[3-[4-(3-hydroxypropoxy)butoxy]propoxy]acetate (19i, 230
mg, 0.66 mmol), toluene sulfonyl chloride (189 mg, 0.99 mmol),
triethylamine (134 mg, 1.32 mmol), and DMAP (16 mg) in
dichloromethane (5 mL) was stirred at rt for 4 h. The reaction
mixture was diluted with dichloromethane (100 mL) and washed with
water and brine. The organic layer was dried over sodium sulfate,
filtered, and concentrated. The residue was purified by silica gel
column chromatography using 30% EtOAc in hexanes as eluent to
afford tert-butyl 2-[3-(4-{3-[(4-methylbenzenesulfonyl)oxy]propoxy}-
butoxy)propoxy]acetate (20i) as a colorless oil (250 mg, 73% yield).
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,4,7,10-tetraoxadodecan-12-amido)-3,3-dimethylbutanoyl]-4-hy-
droxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-
1
carboxamide (3e). H NMR (400 MHz, CD3OD): δ 1.06 (s, 9H),
1.80−2.23 (m, 11H), 2.48 (s, 3H), 2.86−2.91 (2s, 3H), 3.26−3.30 (m,
1H), 3.40−3.45 (m, 3H), 3.71−3.73 (m, 8H), 3.81−3.88 (m, 4H),
4.04−4.11 (m, 4H), 4.34−4.38 (m, 1H), 4.52−4.59 (m, 3H), 4.70−
4.73 (m, 1H), 6.88−6.91 (m, 2H), 7.40−7.49 (m, 6H), 7.66−7.69 (2
br s, 1H, exchanged H), 7.86−7.89 (2s, 1H), 8.87 (s, 1H). LC-MS
(ES+): m/z 1036.1/1038.1 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,6,9,12-tetraoxatetradecan-14-amido)-3,3-dimethylbutanoyl]-4-
hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
+
LC-MS (ES+): m/z 492.2 [M + NH4 ]. This material (105 mg, 0.22
1
pyrrolidine-2-carboxamide (3g). H NMR (400 MHz,CD3OD): δ
mmol) was mixed with 10 (80 mg, 0.18 mmol), cesium carbonate
(120 mg, 0.37 mmol) in N,N-dimethylformamide (5 mL), and the
mixture was stirred at 80 °C overnight. The mixture was diluted with
water (20 mL) and extracted with ethyl acetate (50 mL × 2). The
combined organic layers were washed with brine (50 mL × 2) and
dried over anhydrous sodium sulfate. The residue was purified by silica
gel column chromatography using ethyl acetate/petroleum ether (7/3)
as eluent to afford tert-butyl 1-{4-[(5-bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-oate (23i) as a black oil (100 mg, 74%
yield). LC-MS (ES+): m/z 736.4/738.4 [M + H+]. The tert-butyl ester
23i (100 mg, 0.14 mmol) in dichloromethane (5 mL) and
trifluoroacetic acid (3 mL) was stirred at rt for 2 h. The reaction
mixture was concentrated under reduced pressure to provide the
corresponding carboxylic acid 24i as a black oil (92 mg, 100% yield).
LC-MS (ES+): m/z 680.3/682.3 [M + H+]. This carboxylic acid 24i
(100 mg, 0.147 mmol) was mixed with 18f (68 mg, 0.147 mmol) and
DIPEA (77 mg, 0.6 mmol) in dry DMF (3 mL), and HATU (114 mg,
0.3 mmol) was added at 0 °C. The resulting mixture was stirred at rt
for 0.5 h. The mixture was partitioned between ethyl acetate and
water. The organic phase was washed with water and brine and dried
over anhydrous sodium sulfate, filtered, and concentrated. The residue
was purified by preparative TLC to afford 3i as a white solid (22.5 mg,
15% yield). 1H NMR (400 MHz, CD3OD): δ 1.05 (s, 9H), 1.60−1.64
(m, 4H), 1.80−2.26 (m, 15H), 2.48 (s, 3H), 2.86−2.91 (2s, 3H),
3.26−3.28 (m, 1H), 3.40−3.54 (m, 9H), 3.59−3.64 (m, 4H), 3.83−
3.88 (m, 2H), 3.97−4.06 (m, 4H), 4.34−4.38 (m, 1H), 4.52−4.61 (m,
3H), 4.71−4.72 (m, 1H), 6.87−6.90 (m, 2H), 7.41−7.49 (m, 6H),
7.87−7.90 (2s, 1H), 8.88 (s, 1H). LC-MS (ES+): m/z 1092.5 and
1094.5 [M + H+].
PROTACs Prepared According to Method A As Described in
Scheme 3: 3b, 3d, 3e, 3g, 3h, 3k, and 3n. (2S,4R)-1-[(2S)-2-{2-[2-
(2-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-methylformamido)propyl]-
amino}pyrimidin-2-yl)amino]phenoxy}ethoxy)ethoxy]acetamido}-
3,3-dimethylbutanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)-
phenyl]methyl}pyrrolidine-2-carboxamide (3b). 1H NMR (400
MHz, CDCl3): δ 0.95 (s, 9H), 1.74−1.77 (m, 2H), 1.85−1.90 (m,
2H), 1.94−2.01 (m, 1H), 2.14−2.20 (m, 3H), 2.33−2.37 (m, 2H),
2.45−2.54 (m, 4H), 2.89 (s, 3H), 3.25−3.32 (m, 1H), 3.40−3.49 (m,
4H), 3.60−3.73 (m, 5H), 3.84 (d, J = 4.8 Hz, 2H), 3.93−4.02 (m,
2H), 4.10−4.15 (m, 3H), 4.29−4.34 (m, 1H), 4.46−4.58 (m, 3H),
4.74 (t, J = 8.0 Hz, 1H), 6.57 (br, 1H), 6.86−6.88 (m, 2H), 7.25−7.26
(m, 1H), 7.34−7.36 (m, 4H), 7.43−7.45 (m, 2H), 7.56−7.59 (m, 1H),
0.94 (s, 9H), 1.62−1.72 (m, 7H), 1.75−1.96 (m, 4H), 2.08−2.12 (m,
4H), 2.36 (s, 3H), 2.74−2.79 (2s, 3H), 3.14−3.16 (m, 1H), 3.31−3.42
(m, 5H), 3.50−3.60 (m, 8H), 3.68−3.79 (m, 2H), 3.84−3.94 (m, 4H),
4.22−4.24 (m, 1H), 4.40−4.47 (m, 3H), 4.58−4.60 (m, 1H), 6.73−
6.79 (m, 2H), 7.29−7.36 (m, 6H), 7.74−7.77 (2s, 1H), 8.76 (s, 1H).
LC-MS (ES+): m/z 1064.2 and 1066.2 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,4,7,10,13-pentaoxapentadecan-15-amido)-3,3-dimethylbutano-
yl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3h). H NMR (400 MHz, CD3OD): δ
1.06 (s, 9H), 1.81−1.85 (m, 3H), 2.01−2.26 (m, 8H), 2.50 (s, 3H),
2.85−2.93 (2 br s, 3H), 3.42−3.49 (m, 4H), 3.67−3.87 (m, 15H),
4.01−4.05 (m, 3H), 4.14−4.17 (m, 2H), 4.35−4.39 (m, 1H), 4.52−
4.58 (m, 3H), 4.70−4.73 (m, 1H), 7.03−7.07 (m, 2H), 7.35−7.49 (m,
6H), 7.66−7.68 (2s, 1H, exchanged H), 7.93−7.98 (2 br s, 1H), 8.94
(s, 1H). LC-MS (ES+): m/z 1080.2 and 1082.2 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,4,7,10,13,16-hexaoxaoctadecan-18-amido)-3,3-dimethylbutano-
yl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3k). H NMR (400 MHz, CD3OD): δ
1.06 (s, 9H), 1.80−2.23 (m, 11H), 2.48 (s, 3H), 2.86−2.91 (2s, 3H),
3.26−3.28 (m, 1H), 3.42−3.49 (m, 3H), 3.60−3.70 (m, 17H), 3.79−
3.84 (m, 3H), 3.88−3.90 (m, 1H), 4.04−4.12 (m, 4H), 4.34−4.38 (m,
1H), 4.51−4.54 (m, 1H), 4.58−4.62 (m, 1H), 4.71−4.72 (m, 1H),
6.88−6.92 (m, 2H), 7.41−7.50 (m, 6H), 7.87−7.90 (2s, 1H), 8.88 (s,
1H). LC-MS (ES+): m/z 1124.4 and 1126.4 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,4,7,10,13,16,19-heptaoxahenicosan-21-amido)-3,3-dimethylbu-
tanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3n). H NMR (400 MHz, CD3OD): δ
1.06 (s, 9H), 1.81−1.86 (m, 4H), 2.01−2.04 (m, 3H), 2.18−2.26 (m,
4H), 2.48 (s, 3H), 2.86−2.91 (2s, 3H), 3.43−3.46 (m, 4H), 3.60−3.69
(m, 20H), 3.83−3.85 (m, 4H), 4.04−4.11 (m, 4H), 4.34−4.38 (m,
1H), 4.52−4.59 (m, 3H), 4.71−4.72 (m, 1H), 6.90−6.93 (m, 2H),
7.41−7.51 (m, 6H), 7.87−7.90 (2s, 1H), 8.89 (s, 1H). LC-MS (ES+):
m/z 1168.4 and 1170.4 [M + H+].
PROTACs Prepared According to Method B As Described in
Scheme 3: 3c, 3f, 3j, 3l, 3m, 3o, and 3p. (2S,4R)-1-[(2S)-2-[6-(2-{4-
[(5-Bromo-4-{[3-(1-cyclobutyl-N-methylformamido)propyl]amino}-
pyrimidin-2-yl)amino]phenoxy}ethoxy)hexanamido]-3,3-dimethyl-
butanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]-
J
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
methyl}pyrrolidine-2-carboxamide (3c). 1H NMR (400 MHz,
CDCl3): δ 0.95 (s, 9H), 1.74−1.77 (m, 2H), 1.85−1.90 (m, 2H),
1.94−2.01 (m, 1H), 2.14−2.20 (m, 3H), 2.33−2.37 (m, 2H), 2.45−
2.54 (m, 4H), 2.89 (s, 3H), 3.25−3.32 (m, 1H), 3.40−3.49 (m, 4H),
3.60−3.73 (m, 5H), 3.84 (d, J = 4.8 Hz, 2H), 3.93−4.02 (m, 2H),
4.10−4.15 (m, 3H), 4.29−4.34 (m, 1H), 4.46−4.58 (m, 3H), 4.74 (t, J
= 8.0 Hz, 1H), 6.57 (br, 1H), 6.86−6.88 (m, 2H), 7.25−7.26 (m, 1H),
7.34−7.36 (m, 4H), 7.43−7.45 (m, 2H), 7.56−7.59 (m, 1H), 7.87−
7.93 (2s, 1H), 8.78 (s, 1H). LC-MS (ES+): m/z 1004.4 and 1006.4 [M
+ H+].
(2S,4R)-1-[(2S)-2-(2-{3-[(5-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenoxy}-
pentyl)oxy]propoxy}acetamido)-3,3-dimethylbutanoyl]-4-hydroxy-
N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-car-
boxamide (3f). 1H NMR (400 MHz, DMSO-d6): δ 8.98 (m, 2H), 8.60
(m, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 7.6 Hz, 2H), 7.35−7.40
(m, 5H), 7.05−6.95 (m, 1H), 6.85−6.82 (m, 2H), 5.15 (s, 1H), 4.58−
4.21 (m, 5H), 3.92−3.88 (m, 4H), 3.69−3.41 (m, 6H), 3.37 (m, 5H),
3.19 (m, 1H), 2.82−2.75 (2s, 3H), 2.45 (s, 3H), 2.20−1.65 (m, 15H),
1.58−1.50 (m, 2H), 1.47−1.40 (m, 2H), 0.94 (s, 9H). LC-MS (ES+):
m/z 1048.2 and 1050.2 [MH+].
2.29−2.25 (m, 4H), 2.20−1.98 (m, 3H), 1.94−1.75 (m, 5H), 1.74−
1.58 (m, 20H), 1.57−1.43 (m, 2H), 1.34−1.32 (m, 1H), 1.06 (s, 9H).
LC-MS (ES+): m/z 1292.7 and 1294.7 [M + H+].
(2S,4S)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbutanoyl]-4-
hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
pyrrolidine-2-carboxamide (4). This compound was prepared using a
procedure similar to that for compound 3i. 1H NMR (400 MHz,
CD3OD): δ 8.88 (s, 1H), 7.87 (2s, 1H), 7.49−7.40 (m, 6H), 6.90−
6.86 (m, 2H), 4.63 (s, 1H), 4.58−4.54 (m, 2H), 4.39−4.35 (m, 2H),
4.06−3.96 (m, 5H), 3.75−3.70 (m, 1H), 3.63−3.52 (m, 4H), 3.50−
3.42 (m, 11H), 3.32−3.28 (m, 1H), 2.91 (s, 2H), 2.86 (s, 1H), 2.49−
2.48 (m, 4H), 2.25−2.10 (m, 3H), 2.02−1.99 (m, 4H), 1.89−1.82 (m,
5H), 1.63−1.60 (m, 4H), 1.05 (s, 9H). LC-MS (ES+): m/z 1091.8,
1093.8 [M + H+].
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbutanoyl]-4-
hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
pyrrolidine-2-carboxamide (5a). A solution of 20i (1.0 g, 2.11 mmol)
in dichloromethane (10 mL) and trifluoroacetic acid (5.0 mL) was
stirred at rt for 3 h. The resulting mixture was concentrated under
reduced pressure to give the corresponding carboxylic acid as a light
yellow oil (884 mg). This oil (884 mg, 2.11 mmol) was mixed with 18f
(1.18 g, 2.74 mmol) and DIPEA (1.36 g, 10.5 mmol) in N,N-
dimethylformamide (10 mL), and HATU (962 mg, 2.53 mmol) was
added at 0 °C. The resulting mixture was stirred at rt for 2 h. The
mixture was diluted with water (50 mL) and extracted with ethyl
acetate (50 mL × 3). The combined organic layers were washed with
brine (50 mL × 3), dried over anhydrous sodium sulfate, and
evaporated under reduced pressure. The residue was purified by silica
gel column chromatography using dichloromethane/methanol (10/1)
as eluent to give 28 as a pale yellow oil (1.0 g, 57% yield). This oil
(100 mg, 0.12 mmol) was mixed with N-[3-([2-[(4-hydroxyphenyl)-
amino]pyrimidin-4-yl]amino)propyl]-N-methylcyclobutanecarboxa-
mide (29a, 43 mg, 0.12 mmol, prepared from the hydrogenation of 10
in the presence of palladium on carbon) and cesium carbonate (78 mg,
0.24 mmol) in N,N-dimethylformamide (5 mL), and the mixture was
stirred at 80 °C for 5 h. The resulting solution was diluted with water
(30 mL) and extracted with ethyl acetate (50 mL × 3). The combined
organic layers were washed with brine (50 mL × 3), dried over
anhydrous sodium sulfate, and concentrated under reduced pressure.
The residue was purified by preparative HPLC (XBridge Shield RP18
OBD Column, 5 μm, 19 × 150 mm; mobile phase, water with 10
mmol/L ammonium bicarbonate and acetonitrile, held in 73.0%
acetonitrile for 11 min; detector, UV 254 nm) to afford 5a as an off-
white solid (26 mg, 21% yield). 1H NMR (400 MHz, CD3OD): δ 8.88
(s, 1H), 7.70−7.60 (m, 1H), 7.47−7.39 (m, 6H), 6.86−6.84 (m, 2H),
5.88−5.80 (m, 1H), 4.68 (s, 1H), 4.60−4.52 (m, 3H), 4.35−4.30 (m,
1H), 4.03−4.02 (m, 2H), 3.96−3.94 (d, J = 4.4 Hz, 2H), 3.87−3.80
(m, 2H), 3.61−3.50 (m, 5H), 3.44−3.29 (m, 9H), 2.90 (s, 2H), 2.85
(s, 1H), 2.47 (s, 3H), 2.25−2.10 (m, 4H), 2.00−1.96 (m, 5H), 1.86−
1.80 (m, 6H), 1.60−1.57 (m, 4H), 1.03 (s, 9H). LC-MS (ES+): m/z
1014.2 [M + H+].
(2S,4R)-1-[(2S)-2-{6-[4-(4-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenoxy}-
butoxy)butoxy]hexanamido}-3,3-dimethylbutanoyl]-4-hydroxy-N-
{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
1
mide (3j). H NMR (300 MHz, CD3OD): δ 8.87 (s, 1H), 7.87 (2s,
1H), 7.47−7.39 (m, 6H), 6.89−6.85 (m, 2H), 4.63 (s, 1H), 4.57−4.50
(m, 3H), 4.35 (d, J = 15.3 Hz, 1H), 4.00−3.79 (m, 4H), 3.52−3.31
(m, 12H), 3.30−3.27 (m, 1H), 2.89−2.85 (2s, 3H), 2.48 (s, 3H),
2.28−2.04 (m, 9H), 1.85−1.74 (m, 7H), 1.63−1.54 (m, 8H), 1.39−
1.37 (m, 2H), 1.04 (s, 9 H). LC-MS (ES+): m/z 1104.10 and 1106.10
[M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,4,9,14,17-pentaoxanonadecan-19-amido)-3,3-dimethylbutano-
yl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3l). H NMR (300 MHz, CD3OD): δ
8.87 (s, 1H), 7.87 (2s, 1H), 7.49−7.39 (m, 6H), 6.91−6.88 (m, 2H),
4.69 (s, 1H), 4.61−4.50 (m, 3H), 4.37−4.32 (m, 1H), 4.09−4.03 (m,
4H), 3.90−3.31 (m, 21H), 3.27−3.24 (m, 1H), 2.90−2.85 (2s, 3H),
2.47 (s, 3H), 2.25−1.97 (m, 6H), 1.86−1.77 (m, 3H), 1.62 (br, 8H),
1.04 (s, 9H). LC-MS (ES+): m/z 1136.10 and 1138.10 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,9,13,17-pentaoxaicosan-20-amido)-3,3-dimethylbutanoyl]-4-
hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3m). H NMR (300 MHz, CD3OD): δ
8.87 (s, 1 H), 7.87 (2s, 1H), 7.48−7.39 (m, 6H), 6.89−6.85 (m, 2H),
4.66−4.52 (m, 4H), 4.37 (m, 1H), 4.06−4.02 (m, 2H), 3.87−3.58 (m,
6H), 3.54−3.42 (m, 16H), 3.32−3.30 (m, 1H), 2.90−2.85 (2s, 3 H),
2.48−2.47 (m, 5H), 2.25−1.98 (m, 8H), 1.84−1.72 (m, 10H), 1.03 (s,
9 H). LC-MS (ES+): m/z 1150.1 and 1152.1 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,4,7,12,17,20-hexaoxadocosan-22-amido)-3,3-dimethylbutanoyl]-
4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3o). H NMR (300 MHz, CD3OD): δ
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-fluoropyrimidin-2-yl)amino]-
phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbu-
tanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
pyrrolidine-2-carboxamide (5b). This compound was prepared
8.87 (s, 1H), 7.87 (2s, 1H), 7.49−7.39 (m, 6H), 6.90 (d, J = 11.7 Hz,
2H), 4.69 (s, 1 H), 4.58−4.52 (m, 3 H), 4.37 (m, 1 H), 4.12 (m, 2H),
4.02 (br s, 2H), 3.86−3.81 (m, 4H), 3.70−3.50 (m, 8H), 3.49−3.35
(m, 12H), 3.32−3.31 (m, 1H), 2.90−2.85 (2s, 3 H), 2.48 (s, 3H),
2.22−1.81 (m, 10H), 1.61−1.58 (br, 8H), 1.04 (s, 9 H). LC-MS
(ES+): m/z 1180.2 and 1182.2 [M + H+].
1
according to the procedure for 5a. H NMR (400 MHz, CD3OD):
δ 8.88 (s, 1H), 7.65−7.64 (2s, 1H), 7.48−7.41 (m, 6H), 6.88−6.85
(m, 2H), 4.71 (s, 1H), 4.60−4.54 (m, 3H), 4.38−4.34 (m, 1H), 4.05−
4.02 (m, 2H), 3.98−3.97 (d, J = 4.4 Hz, 2H), 3.87−3.80 (m, 2H),
3.64−3.53 (m, 4H), 3.48−3.42 (m, 10H), 3.33−3.32 (m, 1H), 2.92 (s,
2H), 2.87 (s, 1H), 2.49 (s, 3H), 2.25−2.19 (m, 4H), 2.03−1.85 (m,
10H), 1.63−1.60 (m, 4H), 1.05 (s, 9H). LC-MS (ES+): m/z 1032.1
[M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Chloro-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbutanoyl]-4-
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,7,12,17,22,28-hexaoxatriacontan-30-amido)-3,3-dimethylbuta-
noyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (3p). H NMR (400 MHz, CD3OD): δ
8.89 (s, 1H), 7.91 (br, 1H), 7.48−7.42 (m, 6H), 6.90−6.86 (m, 2H),
4.71 (s, 1H), 4.63−4.51 (m, 3H), 4.41−4.36 (m, 1H), 4.03−3.95 (m,
4H), 3.94−3.79 (m, 2H), 3.56 (t, J = 6.0 Hz, 2H), 3.49−3.37 (m,
20H), 3.32−3.27 (m, 1H), 2.91 (s, 2H), 2.86 (s, 1H), 2.50 (s, 3H),
K
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
resulting solution was diluted with water (20 mL) and extracted with
ethyl acetate (50 mL × 3). The combined organic layers were washed
with brine (50 mL × 3) and dried over anhydrous sodium sulfate. The
residue was purified by silica gel column chromatography using ethyl
acetate/petroleum ether (9/1) as eluent to provide N-[3-({5-
cyclobutyl-2-[(4-hydroxyphenyl)amino]pyrimidin-4-yl}amino)-
propyl]-N-methylcyclobutanecarboxamide as a black oil (70.0 mg, 64%
yield). This material was used for the preparation of 5h with the same
1
pyrrolidine-2-carboxamide (5c). H NMR (400 MHz, CD3OD): δ
8.88 (s, 1H), 7.80−7.77 (2s, 1H), 7.49−7.41 (m, 6H), 6.90−6.86 (m,
2H), 4.71 (s, 1H), 4.58−4.54 (m, 3H), 4.37 (s, 1H), 4.06−4.03 (m,
2H), 3.98−3.97 (d, J = 4.8 Hz, 2H), 3.87−3.80 (m, 2H), 3.62−3.51
(m, 4H), 3.50−3.32 (m, 10H), 3.29−3.20 (m, 1H), 2.91 (s, 2H), 2.86
(s, 1H), 2.49 (s, 3H), 2.26−1.99 (m, 9H), 1.89−1.83 (m, 5H), 1.63−
1.60 (m, 4H), 1.05 (s, 9H). LC-MS (ES+): m/z 1048.1, 1050.1 [M +
H+].
1
method as that described for 5a. H NMR (400 MHz, CD3OD): δ
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-iodopyrimidin-2-yl)amino]-
phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbu-
tanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
8.88 (s, 1H), 7.64 (2s, 1H), 7.49−7.46 (m, 4H), 7.43−7.41 (d, J = 8.0
Hz, 2H), 6.88−6.85 (m, 2H), 4.71 (s, 1H), 4.60−4.50 (m, 3H), 4.38
(m, 1H), 4.05−3.97 (m, 4H), 3.95−3.75 (m, 2H), 3.63−3.59 (m, 7H),
3.53−3.41 (m, 8H), 3.30−3.20 (m, 1H), 2.91 (s, 2H), 2.85 (s, 1H),
2.49 (s, 3H), 2.48−2.40 (m, 2H), 2.25−1.98 (m, 12H), 1.79−1.90 (m,
6H), 1.63−1.60 (m, 4H), 1.05 (s, 9H). LC-MS (ES+): m/z 1068.4 [M
+ H+].
1
pyrrolidine-2-carboxamide (5d). H NMR (400 MHz, CD3OD): δ
8.88 (s, 1H), 8.02 (2s, 1H), 7.48−7.40 (m, 6H), 6.89−6.86 (m, 2H),
4.71 (s, 1H), 4.60−4.53 (m, 3H), 4.37 (s, 1H), 4.06−4.03 (m, 2H),
3.97−3.87 (m, 4H), 3.62−3.50 (m, 4H), 3.48−3.32 (m, 10H), 3.30−
3.20 (m, 1H), 2.90 (s, 2H), 2.85 (s, 1H), 2.49 (s, 3H), 2.24−1.99 (m,
9H), 1.88−1.81 (m, 5H), 1.62−1.59 (m, 4H), 1.05 (s, 9H). LC-MS
(ES+): m/z 1140.4 [M + H+].
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-(trifluoromethyl)pyrimidin-2-
yl)amino]phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-di-
methylbutanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)-
phenyl]methyl}pyrrolidine-2-carboxamide (5e). 1H NMR (400
MHz, CD3OD): δ 8.88 (s, 1H), 8.02 (2s, 1H), 7.51−7.41 (m, 6H),
6.92−6.89 (m, 2H), 4.71 (s, 1H), 4.58−4.50 (m, 3H), 4.38 (s, 1H),
4.07−4.05 (m, 2H), 3.98−3.80 (m, 4H), 3.63−3.59 (m, 4H), 3.53−
3.32 (m, 10H), 3.30−3.20 (m, 1H), 2.89 (s, 2H), 2.84 (s, 1H), 2.48 (s,
3H), 2.24−1.99 (m, 9H), 1.89−1.81 (m, 5H), 1.63−1.60 (m, 4H),
1.05 (s, 9H). LC-MS (ES+): m/z 1083.1 [M + H+].
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-ethenylpyrimidin-2-yl)amino]-
phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbu-
tanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (5i). H NMR (400 MHz, CD3OD): δ
1.04 (s, 9H), 1.60−1.63 (m, 4H), 1.80−2.28 (m, 15H), 2.49 (s, 3H),
2.85−2.91 (2s, 3H), 3.40−3.65 (m, 14H), 3.80−4.08 (m, 6H), 4.34−
4.40 (m, 1H), 4.49−4.62 (m, 3H), 4.71 (s, 1H), 5.22−5.28 (m, 1H),
5.51−5.60 (m, 1H), 6.59−6.66 (m, 1H), 6.90−6.93 (m, 2H), 7.41−
7.50 (m, 6H), 7.82−7.88 (2s, 1H), 8.89 (s, 1H). LC-MS (ES+): m/z
1040.56 1041.0 [M + H+].
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-cyclopropylpyrimidin-2-yl)-
amino]phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dime-
thylbutanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]-
methyl}pyrrolidine-2-carboxamide (5j). 1H NMR (400 MHz,
CD3OD): δ 0.57−0.59 (m, 2H), 1.01−1.05 (m, 11H), 1.61−1.63
(m, 5H), 1.84−2.26 (m, 15H), 2.49 (s, 3H), 2.87, 2.94 (2s, 3H),
3.45−3.65 (m, 14H), 3.80−3.99 (m, 4H), 4.09 (t, J = 6.2 Hz, 2H),
4.35−4.39 (m, 1H), 4.54−4.71 (m, 4H), 6.98−7.01 (m, 2H), 7.35−
7.49 (m, 7H), 8.89 (s, 1H). LC-MS (ES+): m/z 1054.58 1054.9 [M +
H+].
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-methylpyrimidin-2-yl)amino]-
phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbu-
tanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
pyrrolidine-2-carboxamide (5f). H NMR (400 MHz, CD3OD): δ
1.04 (s, 9H), 1.61−1.65 (m, 4H), 1.77−2.28 (m, 18H), 2.49 (s, 3H),
2.86−2.92 (2s, 3H), 3.38−3.65 (m, 14H), 3.80−4.10 (m, 6H), 4.35−
4.71 (m, 5H), 6.94−6.98 (m, 2H), 7.38−7.52 (m, 7H), 8.90 (s, 1H).
LC-MS (ES+): m/z 1028.3 [M + H+].
PROTACs 6a−e Prepared According to the Procedure for 3i.
(2S,4R)-1-[2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)acetyl]-4-hydroxy-N-{[4-(4-
methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide
( 2 S , 4 R ) - 1 - [ ( 2 S ) - 2 - ( 1 - { 4 - [ ( 4 - { [ 3 - ( 1 - C y c l o b u t y l - N -
methylformamido)propyl]amino}-5-ethylpyrimidin-2-yl)amino]-
phenyl}-1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbu-
tanoyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
1
(6a). H NMR (400 MHz, CD3OD): δ 8.87 (s, 1H), 7.88 and 7.85
1
pyrrolidine-2-carboxamide (5g). H NMR (400 MHz, CD3OD): δ
(2s, 1H), 7.41−7.47 (m, 6H), 6.88 (dd, J = 8.8 Hz, 1.8 Hz, 2H), 4.58
(t, J = 7.9 Hz, 1H), 4.36−4.54 (m, 4H), 4.11−4.19 (m, 1H), 4.01−
4.08 (m, 3H), 3.96 (d, J = 2.0 Hz, 2H), 3.76 (dd, J = 10.9 Hz, 4.2 Hz,
1H), 3.55−3.63 (m, 4H), 3.36−3.52 (m, 9H), 3.22−3.29 (m, 1H),
2.89 (s, 2H), 2.84 (s, 1H), 2.47 (s, 3H), 2.15−2.32 (m, 5H), 2.03−
2.14 (m, 2H), 1.94−2.02 (m, 3H), 1.78−1.88 (m, 5H), 1.54−1.63 (m,
4H). LC-MS (ES+): m/z 1036.3, 1038.3 [M + H+].
1.04 (s, 9H), 1.21−1.26 (m, 3H), 1.61−1.63 (m, 4H), 1.84−2.28 (m,
15H), 2.43−2.50 (m, 5H), 2.86−2.92 (2s, 3H), 3.41−3.65 (m, 14H),
3.80−3.91 (m, 2H), 3.94−4.03 (m, 2H), 4.07−4.12 (m, 2H), 4.35−
4.40 (m, 1H), 4.49−4.62 (m, 3H), 4.71−4.80 (m, 1H), 6.98−7.00 (m,
2H), 7.36−7.49 (m, 7H), 8.89 (s, 1H). LC-MS (ES+): m/z 1043.1 [M
+ H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Cyclobutyl-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)-3,3-dimethylbutanoyl]-4-
hydroxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}-
pyrrolidine-2-carboxamide (5h). A mixture of N-[3-[(5-bromo-2-
chloropyrimidin-4-yl)amino]propyl]-N-methylcyclobutanecarboxa-
mide (9, 360 mg, 1.00 mmol), cyclobutylboronic acid (300 mg, 3.00
mmol), sodium carbonate (212.0 mg, 2.00 mmol), and 1,1′-
bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichlorome-
thane complex (73 mg, 0.10 mmol) in toluene/water (5/1) (10 mL)
was stirred at 100 °C under nitrogen atmosphere for 3 h. The resulting
solution was extracted with ethyl acetate (50 mL × 3), and the
combined organic layers were washed with brine (50 mL × 3) and
dried over anhydrous sodium sulfate. The residue was purified by silica
gel column chromatography using ethyl acetate/petroleum ether (1/1)
as eluent to give N-[3-[(5-cyclobutyl-2-chloropyrimidin-4-yl)amino]-
propyl]-N-methylcyclobutanecarboxamide as a light-yellow oil (100
mg, 30% yield). LC-MS (ES+): m/z 337.1, 339.1 [M + H+]. This oily
material (90 mg, 0.27 mmol) was mixed with 4-aminophenol (58 mg,
0.54 mmol) and 4-methylbenzenesulfonic acid (23 mg, 0.13 mmol) in
dioxane (10 mL), and the mixture was stirred at 90 °C overnight. The
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)propanoyl]-4-hydroxy-N-
{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
1
mide (6b). H NMR (400 MHz, CD3OD): δ 8.87 (s, 1H), 7.88 and
7.85 (2s, 1 H), 7.31−7.54 (m, 6H), 6.89 (d, J = 9.00 Hz, 2H), 4.74 (q,
J = 6.98 Hz, 1H), 4.55−4.64 (m, 1H), 4.35−4.54 (m, 3H), 4.04 (t, J =
6.16 Hz, 2H), 3.90−3.97 (m, 2H), 3.73−3.82 (m, 2H), 3.55−3.64 (m,
4H), 3.38−3.54 (m, 9H), 3.22−3.29 (m, 1H), 2.90 and 2.84 (2s, 3H),
2.48 (s, 3H), 2.13−2.29 (m, 5H), 1.94−2.11 (m, 5H), 1.76−1.90 (m,
5H), 1.56−1.63 (m, 4H), 1.32 (d, J = 6.5 Hz, 3H). LC-MS (ES+): m/z
1050.3, 1052.3 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)butanoyl]-4-hydroxy-N-
{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
1
mide (6c). H NMR (400 MHz, CD3OD): δ 8.87 (s, 1H), 7.88 and
7.86 (2s, 1H), 7.37−7.48 (m, 6H), 6.89 (d, J = 9.0 Hz, 2H), 4.70 (dd, J
= 7.1, 5.6 Hz, 1H), 4.59 (t, J = 8.2 Hz, 1H), 4.43−4.53 (m, 2H), 4.36−
4.42 (m, 1H), 4.04 (t, J = 6.3 Hz, 2H), 3.92−3.98 (m, 2H), 3.79 (d, J =
2.5 Hz, 2H), 3.56−3.64 (m, 4H), 3.47−3.55 (m, 3H), 3.37−3.47 (m,
L
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6H), 3.19−3.29 (m, 1H), 2.90 (s, 2H), 2.84 (s, 1H), 2.48 (s, 3H),
2.14−2.30 (m, 5H), 1.96−2.12 (m, 5H), 1.65−1.95 (m, 7H), 1.59 (dt,
J = 5.8, 3.0 Hz, 4H), 0.93−0.97 (t, J = 7.0 Hz, 3H). LC-MS (ES+): m/z
1064.3, 1066.3 [M + H+].
purified by silica gel column chromatography using ethyl acetate/
petroleum ether (1:5) as eluent to afford 10 as a brown solid (1.52 g,
63% yield). 1H NMR (300 MHz, d-DMSO): δ 8.96 (2s, 1 H), 8.86 (s,
1 H), 7.93 (2s, 1 H), 7.44 (d, J = 8.7 Hz, 2 H), 6.97−6.87 (m, 1 H),
6.66 (d, J = 11.1 Hz, 2 H), 3.37−3.23 (m, 4 H), 3.20−3.15 (m, 1 H),
2.82 (s, 2 H), 2.75 (s, 1 H), 2.17−2.04 (m, 3 H), 1.98−1.67 (m, 5 H).
LC-MS (ES+): m/z 434.1 and 436.1 [M + H+].
4-(4-Methylthiazol-5-yl)benzonitrile (12). A mixture of 4-bromo-
benzonitrile (19.0 g, 104 mmol), 4-methylthiazole (21.0 g, 212 mmol),
KOAc (20.5 g, 244 mmol), and Pd(OAc)2 (700 mg, 3.1 mmol) in
DMA (200 mL) was purged with N2 at 0 °C for 10 min, then heated at
150 °C for 5 h. After cooling to rt, the mixture was quenched with
water (1 L) and extracted with ethyl acetate (500 mL × 3). The
combined organic layers were washed with brine (500 mL), dried over
anhydrous sodium sulfate, and concentrated in vacuo. The residue was
purified by silica gel column chromatography using ethyl acetate/
petroleum ether (1:1) as eluent to afford 12 as a yellow solid (19.0 g,
91.3% yield). 1H NMR (400 MHz, CDCl3): δ 8.77 (s, 1H), 7.73 (d, J
= 7.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 2.57 (s, 3H). LC-MS (ES+):
m/z 201.1 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)pentanoyl]-4-hydroxy-N-
{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
1
mide (6d). H NMR (400 MHz, CD3OD): δ 8.87 (s, 1H), 7.89 and
7.86 (2s, 1H), 7.40−7.47 (m, 6H), 6.85−6.92 (m, 2H), 4.76 (dd, J =
8.0, 4.9 Hz, 1H), 4.58 (t, J = 8.2 Hz, 1H), 4.49 (d, J = 15.5 Hz, 2H),
4.36−4.43 (m, 1H), 4.04 (t, J = 6.2 Hz, 2H), 3.95 (d, J = 2.2 Hz, 2H),
3.67−3.80 (m, 3H), 3.59 (t, J = 6.3 Hz, 4H), 3.39−3.53 (m, 9H),
3.19−3.25 (m, 1H), 2.90 (s, 2H), 2.84 (s, 1H), 2.47 (s, 3H), 2.15−
2.29 (m, 5H), 1.94−2.13 (m, 5H), 1.76−1.89 (m, 6H), 1.56−1.70 (m,
5H), 1.38 (m, 2H), 0.90−0.95 (m, 3H). LC-MS (ES+): m/z 1078.3,
1080.3 [M + H+].
(2S,4R)-1-[(2S)-2-(1-{4-[(5-Bromo-4-{[3-(1-cyclobutyl-N-
methylformamido)propyl]amino}pyrimidin-2-yl)amino]phenyl}-
1,5,10,14-tetraoxahexadecan-16-amido)-3-methylbutanoyl]-4-hy-
droxy-N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-
1
(4-(4-Methylthiazol-5-yl)phenyl)methanamine (13). To a solution
of 12 (17 g, 85 mmol) in THF (300 mL) was added LiAlH4 (6.34 g,
167 mmol) in batches at 0 °C. The resulting mixture was heated at 70
°C for 5 h. The reaction was quenched by 10% aqueous NaOH
solution at 0 °C and stirred for 30 min. The mixture was filtered
through a pad of Celite, and the filtered cake was washed with 10%
MeOH/CH2Cl2 four times. The filtrate was concentrated under
reduced pressure to afford the desired product 13 (11 g, 63.4% yield),
which was used in the next step without further purification. 1H NMR
(400 MHz, CDCl3): δ 8.67 (s, 1H), 7.29−7.43 (m, 4H), 3.92 (s, 2H),
2.54 (s, 3H), 1.62 (br, 2H). LC-MS (ES+): m/z 205.1 [M + H+].
(2R,4R)-N-(4-(4-Methylthiazol-5-yl)benzyl)-4-hydroxypyrrolidine-
2-carboxamide Hydrochloride (16). To a solution of trans-N-Boc-4-
hydroxyproline (14) (1.9 g, 8.2 mmol), 13 (1.7 g, 8.3 mmol), and
DIPEA (5 mL) in DMF (10 mL) was added HATU (4.8 g, 12.6
mmol) at 0 °C. The resulting mixture was stirred at rt for 2 h and
quenched with water. The mixture was extracted with ethyl acetate (50
mL × 3). The combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and concentrated. The residue was purified
by silica gel column chromatography to afford (2S,4R)-tert-butyl 2-((4-
(4-methylthiazol-5-yl)benzyl)carbamoyl)-4- hydroxypyrrolidine-1-car-
boxylate (15) as an off white solid (1.4 g, 40.8% yield). LC-MS (ES+):
m/z 418.1 [M + H+]. This material (1.4 g, 3.3 mmol) was dissolved in
HCl in methanol (15 mL), and the solution was stirred at rt for 2 h.
The solvent was evaporated under reduced pressure to provide 16
(800 mg), which was used in the next step without further purification.
1H NMR (400 MHz, CD3OD): δ 9.73 (s, 1H), 9.01 (br s, 1H), 7.56
carboxamide (6e). H NMR (400 MHz, CD3OD): δ 8.87 (s, 1H),
7.88 and 7.85 (2s, 1H), 7.40−7.46 (m, 6H), 6.87−6.90 (m, 2H), 4.63
(d, J = 6.7 Hz, 1H), 4.58 (t, J = 8.3 Hz, 1H), 4.47−4.53 (m, 2H),
4.35−4.42 (m, 1H), 4.04 (t, J = 6.2 Hz, 2H), 3.96 (d, J = 4.9 Hz, 2H),
3.67−3.82 (m, 3H), 3.60 (m, 4H), 3.37−3.55 (m, 9H), 3.20−3.25 (m,
1H), 2.84 and 2.90 (2s, 3H), 2.47 (s, 3H), 2.13−2.28 (m, 6H), 2.03−
2.12 (m, 2H), 1.96−2.03 (m, 3H), 1.75−1.88 (m, 5H), 1.59 (td, J =
3.0, 5.8 Hz, 4H), 1.01 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H).
LC-MS (ES+): m/z 1078.3, 1080.3 [M + H+].
N-[3-[(5-Bromo-2-chloropyrimidin-4-yl)amino]propyl]-N-methyl-
cyclobutanecarboxamide (9). To a solution of cyclobutanecarboxylic
acid (2.66 g, 26.6 mmol) in N,N-dimethylformamide (100 mL) were
added DIPEA (6.86 g, 53.1 mmol) and HATU (12.1 g, 31.9 mmol).
After stirring at 0−10 °C for 30 min, tert-butyl N-[3-(methylamino)-
propyl]carbamate (5 g, 26.6 mmol) was added. The resulting solution
was stirred at rt for 12 h. The reaction was quenched with water (500
mL) and extracted with ethyl acetate (3 × 100 mL). The combined
organic layers were washed with water (100 mL) and brine (100 mL).
The mixture was dried over anhydrous sodium sulfate and
concentrated in vacuo. The residue was purified by silica gel column
chromatography using ethyl acetate/petroleum ether (1:1) as eluent to
afford tert-butyl N-[3-(1-cyclobutyl-N-methylformamido)propyl]-
carbamate as a colorless oil (5.9 g, 82% yield). LC-MS (ES+): m/z
271.1 [M + H+]. The oily material (13.0 g, 48.1 mmol) in methanol/
HCl (g) (200 mL) was stirred at rt for 1 h. The resulting mixture was
concentrated in vacuo to provide N-(3-aminopropyl)-N-methylcyclo-
butanecarboxamide hydrochloride (8) as a white solid (9.6 g, 97%
yield). LC-MS (ES+): m/z 171.0 [M + H+]. This hydrochloride salt
(9.6 g, 46.4 mmol) was mixed with 5-bromo-2,4-dichloropyrimidine
(10.5 g, 46.3 mmol) in acetonitrile (250 mL), and DIPEA (18.0 g,
139.3 mmol) was added at 0 °C. The resulting solution was slowly
warmed to rt and stirred for 3 h. The reaction was quenched with
water (50 mL) and extracted with ethyl acetate (3 × 100 mL). The
combined organic layers were washed with brine (500 mL), dried over
anhydrous sodium sulfate, and concentrated in vacuo. The residue was
suspended in 200 mL of ethyl acetate/petroleum ether (1/5, v/v), and
the white solid was collected by filtration (11.3 g, 67% yield). 1H NMR
(400 MHz, CDCl3) δ 8.09 (s, 1 H), 7.20 (b, 1 H), 3.49−3.47 (m, 4
H), 3.46−3.28 (m, 1 H), 2.93 (s, 3H), 2.41−2.31 (m, 2 H), 2.24−2.16
(m, 2 H), 2.05−1.80 (m, 2 H), 1.80−1.70 (m, 2 H). LC-MS (ES+): m/
z 360.9 and 362.9 [M + H+].
(d, J = 7.2 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 4.49−4.61 (m, 4H),
3.42−3.46 (m, 2H), 2.56 (s, 3H), 2.47−2.53 (m, 1H), 2.06−2.12 (m,
1H). LC-MS (ES+): m/z 318.1 [M + H+].
(2S,4R)-1-[(2S)-2-Aminopropanoyl]-4-hydroxy-N-{[4-(4-methyl-
1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide Hydro-
chloride (18b). To a solution of (tert-butoxycarbonyl)-L-alanine
(0.84 g, 4.2 mmol), DIPEA (2.44 g, 18.9 mmol), HATU (1.94 g,
5.1 mmol) in DMF (50 mL) was added 16 (1.5 g, 4.2 mmol). The
resulting solution was stirred at rt for 3 h and quenched with water
(100 mL). The mixture was extracted with ethyl acetate (100 mL × 2).
The combined organic layers were washed with water and brine. The
residue was purified by silica gel column chromatography to afford tert-
butyl N-[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(4-methyl-1,3-thiazol-5-
yl)phenyl]methyl}carbamoyl)pyrrolidin-1-yl]-1-oxopropan-2-yl]-
1
carbamate (17b) as an off white solid (1.45 g, 70% yield). H NMR
N-[3-([5-Bromo-2-[(4-hydroxyphenyl)amino]pyrimidin-4-yl]-
amino)propyl]-N-methylcyclobutanecarboxamide (10). A solution
of 9 (2.0 g, 5.56 mmol), 4-aminophenol (1.2 g, 11.0 mmol), and 4-
methylbenzenesulfonic acid (480 mg, 2.79 mmol) in dioxane (30 mL)
was stirred at 90 °C overnight. The pH value of the solution was
adjusted to 7−8 with aqueous sodium bicarbonate solution, and the
resulting solution was extracted with ethyl acetate (20 mL × 3). The
combined organic layers were washed with brine (20 mL × 2), dried
over anhydrous sodium sulfate, and concentrated. The residue was
(400 MHz, CDCl3): δ 8.68 (s, 1H), 7.37−7.39 (m, 3H), 7.27−7.31
(m, 2H), 5.24 (d, J = 7.2 Hz, 1H), 4.76 (t, J = 7.2 Hz, 1H), 4.59 (br s,
1H), 4.37−4.54 (m, 3H), 3.96 (d, J = 11.2 Hz, 1H), 3.02(s, 1H),
2.52−2.57 (m, 4H), 2.09−2.14 (m, 1H), 1.41 (s, 9H), 1.26 (m, 3H).
LC-MS (ES+) m/z 489.2 [M + H+]. This material (1.45 g, 2.97 mmol)
in HCl/dioxane (20 mL) was stirred at rt for 3 h. The mixture was
concentrated in vacuo, and the solid was washed with dichloromethane
in petroleum ether (1:1) to afford the desired product 18b as an off
M
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
white solid (1.23 g, 98% yield). H NMR (400 MHz, CD3OD): δ
brine (300 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by
silica gel flash column chromatography to give a pale red oil (10 g,
61% yield). This oily material (10 g, 24.0 mmol) in HCl/MeOH (100
mL) was stirred at rt for 2 h. The resulting mixture was concentrated
under reduced pressure to afford (2S,4S)-4-hydroxy-N-[[4-(4-methyl-
1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide hydrochlor-
ide (25) as a yellow solid (4.5 g, 53% yield). LC-MS (ES+): m/z 318.1
[M + H+]. This solid (9.0 g, 25.4 mmol) was mixed with (2S)-2-[(tert-
butoxy)carbonyl]amino-3,3-dimethylbutanoic acid (5.8 g, 25.1 mmol)
and DIPEA (5.0 g, 38.7 mmol) in N,N-dimethylformamide (50 mL),
and HATU (11.6 g, 30.5 mmol) was added at 0 °C. The resulting
solution was stirred at rt for 16 h. The reaction mixture was diluted
with water (200 mL) and extracted with ethyl acetate (300 mL × 3).
The combined organic layers were washed with water (100 mL) and
brine (100 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by
silica gel flash column chromatography using ethyl acetate/petroleum
ether (10/90) as eluent to give tert-butyl N-[(2S)-1-[(2S,4S)-4-
hydroxy-2-([[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl]carbamoyl)-
pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate (26) as a pale
red oil (10 g, 74% yield). LC-MS (ES+): m/z 531.2 [M + H+]. This
oily material (10 g, 18.8 mmol) in HCl/MeOH (20 mL) was stirred at
rt for 2 h. The resulting mixture was concentrated under reduced
10.07 (s, 1H), 7.56 (br s, 4H), 4.63 (t, J = 8.4 Hz, 1H), 4.52−4.56 (m,
2H), 4.42−4.46 (m, 1H), 4.29−4.31 (m, 1H), 3.59−3.76 (m, 2H),
2.62 (s, 3H), 2.31−2.34 (m, 1H), 2.02−2.08 (m, 1H), 1.51 (d, J = 7.2
Hz, 3H). LC-MS (ES+): m/z 389.2 [M + H+].
Intermediates 18a, 18c−f Prepared Using the Same Method
As That Described in 18b. (2S,4R)-1-(2-Aminoacetyl)-4-hydroxy-
N-{[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-car-
1
boxamide hydrochloride (18a). H NMR (400 MHz, CD3OD): δ
9.67 (br s, 1H), 7.52 (s, 4H), 4.53−4.61 (m, 3H), 4.40−4.44 (m, 1H),
3.72−3.96 (m, 4H), 2.57 (s, 3H), 2.20−2.30 (m, 1H), 2.07−2.11 (m,
1H). LC-MS (ES+): m/z 375.1 [MH+].
(2S,4R)-1-[(2S)-2-Aminobutanoyl]-4-hydroxy-N-{[4-(4-methyl-
1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide Hydro-
chloride (18c). 1H NMR (400 MHz, CD3OD): δ: 9.85 (s, 1H),
7.54 (s, 4H), 4.64 (t, J = 8.8 Hz, 1H), 4.42−4−55 (m, 3H), 4.24 (s,
1H), 3.72−3.78 (m, 2H), 2.59 (s, 3H), 2.28−2.33 (m, 1H), 2.06−2.09
(m, 1H), 1.91−1.96 (m, 2H), 1.05−1.07 (m, 3H). LC-MS (ES+): m/z
403.2 [M + H+].
(2S,4R)-1-[(2S)-2-Aminopentanoyl]-4-hydroxy-N-{[4-(4-methyl-
1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide Hydro-
1
chloride (18d). H NMR (400 MHz, CD3OD): δ 9.97 (s, 1H), 7.55
(s, 4H), 4.64 (t, J = 8.8 Hz, 1H), 4.47−4.55 (m, 3H), 4.26 (s, 1H),
3.72 (m, 2H), 2.61 (s, 3H), 2.28−2.33 (m, 1H), 2.04−2.08 (m,
1H),1.80−1.89 (m, 2H), 1.52−1.58 (m, 2H), 1.00 (t, J = 6.8 Hz, 3H).
LC-MS (ES+): m/z 417.2 [M + H+].
1
pressure to afford 27 as a yellow solid (6 g, 68% yield). H NMR
(DMSO-d6): δ 9.04 (s, 1H), 8.80−8.76 (m, 1H), 8.20 (br, 3H), 7.43−
7.40 (m, 4H), 4.47−4.40 (m, 2H), 4.31−4.22 (m, 2H), 4.09−3.90 (m,
2H), 3.33−3.31 (m, 1H), 2.51 (s, 3H), 2.51−2.49 (m, 1H), 1.80−1.65
(m, 1H), 1.00 (s, 9H). LC-MS (ES+): m/z 431.2 [M + H+].
Western Blot Assay to Determine TBK1/IKKε Degradation
and IRF3 Phosphorylation. Panc02.13 cells were purchased from
ATCC and cultured in RPMI-1640 (Gibco), supplemented with 15%
FBS (ATCC) and 10 units/mL human recombinant insulin (Gibco).
PROTAC treatments were carried out in 12-well plates for 16 h. TLR3
agonist Poly I:C (Invivogen; tlrl-pic) was added for the final 3 h. Cells
were harvested and lysed in RIPA buffer (50 mM Tris at pH 8, 150
mM NaCl, 1% Tx-100, 0.1% SDS, and 0.5% sodium deoxycholate)
supplemented with protease and phosphatase inhibitors. Lysates were
clarified at 16,000g for 10 min, and supernatants were separated by
SDS−PAGE. Immunoblotting was performed using standard proto-
cols. The antibodies used were TBK1 (Cell Signaling#3504), pIRF3
(abcam#ab76493), and GAPDH (Cell Signaling#5174).
Proliferation Assay. The indicated lung cancer cell lines were
plated in 50 μL in 96-well plates at 5000 cells/well. After 24 h, 50 μL
of 2× drug solution (3-fold dilutions, 10 μM maximum concentration)
was added for a final DMSO concentration of 0.1%, and plates were
incubated for 72 h, following which 100 μL of CellTiterGlo reagent
(Promega #G7570) was added to each well, and luminescence was
measured.
IKKε Knockdown. Three unique Trilencer siRNA duplexes were
purchased from Origene with the following sequence information:
siRNA1, AGAUUCACAAGCUGGAUAAGGUGdAdA; siRNA2,
ACCUU CAAGAAGUGGAAUAAAU GdUdG ; siRNA3,
GACAGAAAGCAUAACAUACACUCdGdC. The siRNA sequences
were transfected into Panc02.13 cells in a 12-well plate using
Lipofectamine RNAimax (Invitrogen) following the manufacturer’s
protocols. Cells were harvested 60 h after transfection and analyzed for
protein levels by immunoblotting.
TBK1 Kd Determination. Compounds were shipped as 10 mM
DMSO stocks to DiscoverX (San Diego, CA) to evaluate Kd values in
duplicate against either TBK1 or IKKε in 12-point dose−response
curves starting at the highest concentration of 10 μM.
(2S,4R)-1-[(2S)-2-Amino-3-methylbutanoyl]-4-hydroxy-N-{[4-(4-
methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxamide
1
Hydrochloride (18e). H NMR (400 MHz, CD3OD): δ 9.97 (s, 1H),
7.55 (s, 4H), 4.42−4.67 (m, 4H), 4.13−4.14 (m, 1H), 3.66−3.75 (m,
2H), 2.61 (s, 3H), 2.29−2.34 (m, 2H), 2.03−2.10 (m, 1H), 1.14−1.18
(m, 6H). LC-MS (ES+): m/z 417.2 [M + H+].
(2S,4R)-1-[(2S)-2-Amino-3,3-dimethylbutanoyl]-4-hydroxy-N-{[4-
(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
1
mide Hydrochloride (18f). H NMR (400 MHz, CD3OD): δ 9.84−
9.82 (s, 1H), 7.58−7.54 (m, 4H), 4.71−4.41 (m, 4H), 4.13−4.08 (m,
1H), 3.86−3.71 (m, 2H), 2.60−2.58 (s, 3H), 2.35−2.07 (m, 2H),
1.19−1.12 (m, 9H). LC-MS (ES+): m/z 431.2 [M + H+].
tert-Butyl 2-(4-(tosyloxy)butoxy)acetate (20a). A mixture of tert-
butyl 2-(4-hydroxybutoxy)acetate (19a, 500 mg, 2.5 mmol), 4-
toluenesulfonyl chloride (520 mg, 2.7 mmol), triethylamine (1.5 mL,
8.75 mmol), and DMAP (16 mg, 0.14 mmol) in dichloromethane (20
mL) was stirred at rt overnight. The reaction mixture was diluted with
dichloromethane (50 mL) and washed with water and brine. The
organic layer was dried over sodium sulfate, filtered, and concentrated.
The residue was purified by silica gel column chromatography using
10−15% ethyl acetate in hexanes as eluent to afford 20a as a colorless
oil (700 mg, 87% yield). 1H NMR (400 MHz, CDCl3): δ 1.47 (s, 9H),
1.62−1.67 (m, 2H), 1.75−1.82 (m, 2H), 2.45 (s, 3H), 3.47 (t, J = 6.0
Hz, 2H), 3.89 (s, 2H), 4.07 (t, J = 6.4 Hz, 2H), 7.34 (d, J = 8.0 Hz,
2H), 7.78 (d, J = 8.0 Hz, 2H).
tert-Butyl 2-(4-(4-nitrophenoxy)butoxy)acetate (21a). A mixture
of 4-nitrophenol (240 mg, 1.7 mmol), 20a (700 mg, 2.0 mmol), and
K2CO3 (700 mg, 5.1 mmol) in DMF (5 mL) was stirred at 70 °C for
16 h. The mixture was partitioned between ethyl acetate and water.
The organic phase was washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated. The residue was purified by
silica gel column chromatography using 20−50% ethyl acetate in
hexanes as eluent to provide 21a as a light-yellow solid (600 mg, 90%
1
yield). H NMR (400 MHz, CDCl3): δ 1.50 (s, 9H), 1.80−1.87 (m,
2H), 1.95−2.03 (m, 2H), 3.62 (t, J = 6.0 Hz, 2H), 3.99 (s, 2H), 4.14
(t, J = 6.4 Hz, 2H), 6.97 (d, J = 7.2 Hz, 2H), 8.21 (d, J = 7.2 Hz, 2H).
LC-MS (ES+): m/z 348.2 [M + Na+].
His-Tagged VHL Protein and Fluorescein-Labeled HIF-1α
Peptide. VHL was coexpressed and copurified with Elongin C and B
(VCB) as previously described.23 For the fluorescence polarization
assay, a fluorescein labeled HIF-1α peptide (FAM-DEALA-Hyp-
YIPD) was synthesized and purified as previously described.24
VHL Binding Assay Based on Fluorescence Polarization.
Compounds were serially diluted in 100% DMSO and 2 μL transferred
to Corning Costar 384-well black assay plates (Corning, #3575). To
(2S,4S)-1-[(2S)-2-Amino-3,3-dimethylbutanoyl]-4-hydroxy-N-{[4-
(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}pyrrolidine-2-carboxa-
mide Hydrochloride (27). To a solution of cis-N-Boc-proline 24 (9.0
g, 38.9 mmol), 13 (8.0 g, 39.2 mmol), and DIPEA (8.0 g, 61.9 mmol)
in DMF (100 mL) was added HATU (18.0 g, 47.4 mmol). The
resulting mixture was stirred at rt overnight. The solution was diluted
with water (200 mL) and extracted with ethyl acetate (300 mL × 3).
The combined organic layers were washed with water (300 mL) and
N
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the assay plates, 8 μL of 2.5 μM VHL protein in buffer A (50 mM Tris
pH7.5, 200 mM NaCl, 2 mM DTT and 1% DMSO) was added and
incubated with the compound for 10 min. Next, 10 μL of 40 nM
fluorescein labeled HIF-1α peptide (in buffer A) was added to each
well to give a final peptide concentration of 20 nM and a final VHL
concentration of 125 nM. Plates were incubated in the dark for 2 h.
Fluorescence polarization from the assay plates was read using a
Cytation3 plate reader equipped with a FP Green Cube (Biotek
Instruments, Winooski, VT). FP Values were converted to percent
binding based on control wells and IC50 values calculated in GraphPad
Prism using the four-parameter nonlinear regression dose−response
model.
(5) Lebraud, H.; Wright, D. J.; Johnson, C. N.; Heightman, T. D.
Protein degradation by in-cell self-assembly of proteolysis targeting
chimeras. ACS Cent. Sci. 2016, 2 (12), 927−934.
(6) Schiedel, M.; Herp, D.; Hammelmann, S.; Swyter, S.; Lehotzky,
́
A.; Robaa, D.; Olah, J.; Ovadi, J.; Sippl, W.; Jung, M. Chemically
induced degradation of sirtuin 2 (Sirt2) by a proteolysis targeting
chimera (PROTAC) based on sirtuin rearranging ligands (SirReals). J.
Med. Chem.. DOI: 10.1021/acs.jmedchem.6b01872. Published online:
Apr 5, 2017. http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.
6b01872 (accessed Apr 5, 2017).
(7) Schneekloth, J. S., Jr.; Fonseca, F. N.; Koldobskiy, M.; Mandal, A.;
Deshaies, R.; Sakamoto, K.; Crews, C. M. Chemical genetic control of
protein levels: selective in vivo targeted degradation. J. Am. Chem. Soc.
2004, 126, 3748−3754.
(8) Salami, J.; Crews, C. M. Waste disposal - An attractive strategy for
cancer therapy. Science 2017, 355, 1163−1167.
(9) Schneekloth, A. R.; Pucheault, M.; Tae, H. S.; Crews, C. M.
Targeted intracellular protein degradation induced by a small
molecule: en route to chemical proteomics. Bioorg. Med. Chem. Lett.
2008, 18, 5904−5908.
(10) (a) Doak, B. C.; Over, B.; Giordanetto, F.; Kihlberg, J. Oral
druggable space beyond the rule of 5: insights from drugs and clinical
candidates. Chem. Biol. 2014, 21, 1115−1142. (b) Doak, B. D.; Zheng,
J.; Dobritzsch, D.; Kihlberg, J. How beyond rule of 5 drugs and clinical
candidates bind to their targets. J. Med. Chem. 2016, 59, 2312−2327.
(11) Ottis, P.; Crews, C. M. PROTACs: induced protein degradation
as a therapeutic strategy. ACS Chem. Biol. 2017, 12, 892−898.
(12) (a) Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.;
Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M.
Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4.
Chem. Biol. 2015, 22, 755−763. (b) Raina, K.; Lu, J.; Qian, Y.; Altieri,
M.; Gordon, D.; Rossi, A. M. K.; Wang, J.; Chen, X.; Dong, H.; Siu, K.;
Winkler, J. D.; Crew, A. P.; Crews, C. M.; Coleman, K. G. PROTAC-
induced BET protein degradation as a therapy for castration-resistant
prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 7124−7129.
(c) Saenz, D. T.; Fiskus, W.; Qian, Y.; Manshouri, T.; Rajapakshe, K.;
Raina, K.; Coleman, K. G.; Crew, A. P.; Shen, A.; Mill, C. P.; Sun, B.;
Qiu, P.; Kadia, T. M.; Pemmaraju, N.; DiNardo, C.; Kim, M.-S.;
Nowak, A. J.; Coarfa, C.; Crews, C. M.; Verstovsek, S.; Bhalla, K. N.
Novel BET protein proteolysis-targeting chimera exerts superior lethal
activity than bromodomain inhibitor (BETi) against post-myelopro-
liferative neoplasm secondary (s) AML cells. Leukemia. DOI: 10.1038/
leu.2016.393. Published online: Jan 2, 2017. http://www.nature.com/
leu/journal/vaop/ncurrent/full/leu2016393a.html (accessed Jan 2,
2017).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00635.
Synthetic procedures and related analytical data for the
linker intermediates 19a−p (PDF)
Biological data cited in Tables 1, 2, and 3 (CSV)
AUTHOR INFORMATION
Corresponding Authors
*(A.P.C.) E-mail: andy.crew@arvinas.com.
*(C.M.C.) E-mail: craig.crews@yale.edu.
■
ORCID
Craig M. Crews: 0000-0002-8456-2005
Present Address
∥D.V.: Agios Pharmaceuticals, Cambridge, MA.
Notes
The authors declare the following competing financial
interest(s): C.M.C. is a consultant and shareholder of Arvinas,
LLC, which supports research in his lab. A.P.C, K.R., H.D.,
Y.Q., J.W., B.D.H., A.M., C.F., T.N., and K.C. are shareholders
and employees. D.V., K.S., and J.W. are Arvinas shareholders.
ACKNOWLEDGMENTS
■
We thank Suzhou Medinoah Co. Ltd. and Pharmaron for
providing synthetic assistance. C.M.C. gratefully acknowledges
support from the NIH (R35 CA197589).
(13) Bondeson, D. P.; Mares, A.; Smith, I. E.D.; Ko, E.; Campos, S.;
Miah, A. H.; Mulholland, K. E.; Routly, N.; Buckley, D. L.; Gustafson,
J. L.; Zinn, N.; Grandi, P.; Shimamura, S.; Bergamini, G.; Faelth-
Savitski, M.; Bantscheff, M.; Cox, C.; Gordon, D. A.; Willard, R. R.;
Flanagan, J. J.; Casillas, L. N.; Votta, B. J.; den Besten, W.; Famm, K.;
Kruidenier, L.; Carter, P. S.; Harling, J. D.; Churcher, I.; Crews, C. M.
Catalytic in vivo protein knockdown by small-molecule PROTACs.
Nat. Chem. Biol. 2015, 11, 611−617.
ABBREVIATIONS USED
■
DIPEA, diisopropylethylamine; HATU, 1-[bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; DC50, half maximal
degradation concentration; Dmax, maximal degradation effect
(14) (a) Clark, K.; Plater, L.; Peggie, M.; Cohen, P. Use of the
pharmacological inhibitor BX795 to study the regulation and
physiological roles of TBK1 and IkappaB kinase epsilon: a distinct
upstream kinase mediates Ser-172 phosphorylation and activation. J.
Biol. Chem. 2009, 284, 14136−14146. (b) McIver, E. G.; Bryans, J.;
Birchall, K.; Chugh, J.; Drake, T.; Lewis, S. J.; Osborne, J.; Smiljanic-
Hurley, E.; Tsang, W.; Kamal, A.; Levy, A.; Newman, M.; Taylor, D.;
Arthur, J. S. C.; Clark, K.; Cohen, P. Synthesis and structure-activity
relationships of a novel series of pyrimidines as potent inhibitors of
TBK1/IKKε kinases. Bioorg. Med. Chem. Lett. 2012, 22, 7169−7173.
(c) Yu, T.; Yang, Y.; Yin, D. Q.; Hong, S.; Son, Y.-J.; Kim, J.-H.; Cho, J.
Y. TBK1 inhibitors: a review of patent literature (2011 - 2014). Expert
Opin. Ther. Pat. 2015, 25, 1385−1396.
REFERENCES
■
(1) Toure, M.; Crews, C. M. Small molecule PROTACs: new
approaches to protein degradation. Angew. Chem., Int. Ed. 2016, 55,
1966−1973.
(2) Corson, T. W.; Aberle, N.; Crews, C. M. Design and applications
of bifunctional small molecules: why two heads are better than one.
ACS Chem. Biol. 2008, 3, 677−692.
(3) Sakamoto, K. M.; Kim, K. B.; Verma, R.; Ransick, A.; Stein, B.;
Crews, C. M.; Deshaies, R. J. Development of PROTACs to target
cancer-promoting proteins for ubiquitination and degradation. Mol.
Cell. Proteomics 2003, 2, 1350−1358.
(4) Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews,
C. M.; Deshaies, R. J. PROTACs: chimeric molecules that target
protein to the Skp1-Cullin-F box complex for ubiquitination and
degradation. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554−8559.
(15) Barbie, D. A.; Tamayo, P.; Boehm, J. S.; Kim, S. Y.; Moody, S.
E.; Dunn, I. F.; Schinzel, A. C.; Sandy, P.; Meylan, E.; Scholl, C.;
Frohling, S.; Chan, E. M.; Sos, M. L.; Michel, K.; Mermel, C.; Silver, S.
̈
O
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J.; Weir, B. A.; Reiling, J. H.; Sheng, Q.; Gupta, P. B.; Wadlow, R. C.;
Le, H.; Hoersch, S.; Wittner, B. S.; Ramaswamy, S.; Livingston, D. M.;
Sabatini, D. M.; Meyerson, M.; Thomas, R. K.; Lander, E. S.; Mesirov,
J. P.; Root, D. E.; Gilliland, D. G.; Jacks, T.; Hahn, W. C. Systematic
RNA interference reveals that oncogenic KRAS-driven cancers require
TBK1. Nature 2009, 462, 108−112.
(16) Muvaffak, A.; Pan, Q.; Yan, H.; Fernandez, R.; Lim, J.; Dolinski,
B.; Nguyen, T. T.; Strack, P.; Wu, S.; Chung, R.; Zhang, W.; Hulton,
́
C.; Ripley, S.; Hirsch, H.; Nagashima, K.; Wong, K. K.; Janne, P. A.;
Seidel-Dugan, C.; Zawel, L.; Kirschmeier, P. T.; Middleton, R. E.;
Morris, E. J.; Wang, Y. Evaluating TBK1 as a therapeutic target in
cancers with activated IRF3. Mol. Cancer Res. 2014, 12, 1055−1066.
(17) Larabi, A.; Devos, J. M.; Ng, S.-L.; Nanao, M. H.; Round, A.;
Maniatis, T.; Panne, D. Crystal structure and mechanism of activation
of TANK-binding kinase 1. Cell Rep. 2013, 3, 734−746.
(18) Iwai, K.; Yamanaka, K.; Kamura, T.; Minato, N.; Conaway, R.
C.; Conaway, J. W.; Klausner, R. D.; Pause, A. Identification of the von
Hippel-Lindau tumor-suppressor protein as part of an active E3
ubiquitin ligase complex. Proc. Natl. Acad. Sci. U. S. A. 1999, 96,
12436−12441.
(19) Crews, C. M.; Buckley, D.; Ciulli, A.; Jorgensen, W.; Gareiss, P.
C.; Van Molle, I.; Gustafson, J.; Tae, H.-S.; Michel, J.; Hoyer, D. W.;
Roth, A. G.; Harling, J. D.; Smith, I. E. D.; Miah, A. H.; Campos, S. A.;
Le, J. Compounds and Methods for the Enhanced Degradation of
Targeted Proteins and Other Polypeptides by an E3 Ubiquitin Ligase,
Particularly Targeting the von Hippel-Lindau E3 Ubiquitin Ligase
Using Hydroxyproline Analogs to Disrupt the VHL/HIF-1α
Interaction. WO 2013/106643, Jul 18, 2013.
(20) Galdeano, C.; Gadd, M. S.; Soares, P.; Scaffidi, S.; Van Molle, I.;
Birced, I.; Hewitt, S.; Dias, D. M.; Ciulli, A. Structure-guided design
and optimization of small molecules targeting the protein-protein
interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase
and the hypoxia inducible factor (HIF) alpha subunit with in vitro
nanomolar affinities. J. Med. Chem. 2014, 57, 8657−8663.
(21) Zengerle, M.; Chan, K.-K.; Ciulli, A. Selective small molecule
induced degradation of the BET bromodomain protein BRD4. ACS
Chem. Biol. 2015, 10, 1770−1777.
(22) Gadd, M. S.; Testa, A.; Lucas, X.; Chan, K.-K.; Chen, W.;
Lamont, D. J.; Zengerle, M.; Ciulli, A. Structural basis of PROTAC
cooperative recognition for BRD4 selective protein degradation. Nat.
Chem. Biol. 2017, 13, 514−521.
(23) Hutti, J. E.; Porter, M. A.; Cheely, A. W.; Cantley, L. C.; Wang,
X.; Kireev, D.; Baldwin, A. S.; Janzen, W. P. Development of a high-
throughput assay for identifying inhibitors of TBK1 and IKKε. PLoS
One 2012, 7 (7), e41494.
(24) Komander, D. The emerging complexity of protein ubiquitina-
tion. Biochem. Soc. Trans. 2009, 37 (5), 937−953.
P
DOI: 10.1021/acs.jmedchem.7b00635
J. Med. Chem. XXXX, XXX, XXX−XXX