Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

Article abstract of DOI:10.1021/acs.jmedchem.7b01406

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalyt

Full text of DOI:10.1021/acs.jmedchem.7b01406

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Article
Demonstrating In-Cell Target Engagement using
a Pirin Protein Degradation Probe (CCT367766)
Nicola E. A. Chessum, Swee Y. Sharp, John J Caldwell, A Elisa Pasqua, Birgit Wilding, Giampiero
Colombano, Ian Collins, Bugra Ozer, Meirion Richards, Martin G. Rowlands, Mark Stubbs, Rosemary
Burke, P. Craig McAndrew, Paul A. Clarke, Paul Workman, Matthew D. Cheeseman, and Keith Jones
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01406 • Publication Date (Web): 14 Dec 2017
Downloaded from http://pubs.acs.org on December 15, 2017
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Demonstrating InꢀCell Target Engagement using a
Pirin Protein Degradation Probe (CCT367766)
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Nicola E. A. Chessum, Swee Y. Sharp, John J. Caldwell, A. Elisa Pasqua, Birgit Wilding,
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Giampiero Colombano, Ian Collins, Bugra Ozer, Meirion Richards, Martin Rowlands, Mark
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Stubbs, Rosemary Burke, P. Craig McAndrew, Paul A. Clarke, Paul Workman, Matthew D.
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Cheeseman and Keith Jones
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Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London
SW7 3RP, United Kingdom
ABSTRACT: Demonstrating intracellular protein target engagement is an essential step in the
development and progression of new chemical probes and potential small molecule therapeutics.
However, this can be particularly challenging for poorly studied and nonꢀcatalytic proteins, as
robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical
probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we
developed a heterobifunctional protein degradation probe. Focusing on linker design and
physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three
iterations, validating our efficient strategy for degradation probe design against nonꢀvalidated
protein targets.
INTRODUCTION
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Drug discovery is reliant on recombinant proteins and biochemical screens to develop structure
1
activity relationships (SAR) and progress compounds. However, the conditions in biochemical
assays often display little relevance to the intracellular environment, which can result in a failure
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to translate high target affinity to activity within a living cell. Targetꢀproximal biomarker
3
modulation is the most important confirmation of intracellular target engagement.
Unfortunately, this is often not possible in early stage chemical probe or drug discovery projects,
especially against novel biological targets, where poorly understood biology and pharmacology
make it difficult to discover and robustly validate biomarkers; a process that is particularly
challenging for nonꢀcatalytic proteins.
Owing to the importance of confirming intracellular target engagement, several techniques
have been developed. The overꢀexpression of fusion proteins allows for a direct readout of target
4
occupancy. However, the engineered cell lines are often difficult to generate and the protein
label can impact compound binding. The cellular thermal shift assay (CETSA) is a labelꢀfree
5
technique for intracellular target engagement. It exploits compoundꢀinduced stabilization of
6
protein melting temperatures, but CETSA cannot be applied to all targets. Activityꢀbased protein
profiling (ABPP) methods utilize nonꢀselective irreversible covalent ligands, or intracellular
fluorescence polarization probes, combined with intracellular reversible ligand competition, to
7
8,15
study target engagement. Proteolysis targeting chimeras (PROTACs) and specific and nonꢀ
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genetic IAPꢀdependent protein erasers (SNIPERs) are heterobifunctional molecules that induce
rapid and selective protein degradation, via the proteasome, within living cells. One portion of
the molecule engages the target protein while the other, attached via a flexible linker, recruits an
E3 ligase to ubiquitinate the target, marking it for degradation as part of the cullinꢀRING finger
,
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machinery.
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We recently reported the development of a high affinity pirin chemical probe 1 (CCT251236,
SPR K = 44 nM) discovered through a cellꢀbased phenotypic screen for inhibitors of the heat
D
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shock transcription factor 1 (HSF1) stress pathway. Pirin is an ironꢀbinding member of the
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cupin super family of proteins and has been reported as a putative transcription factor
,
1
2 13
regulator
It has no known enzymatic function in mammalian cells, no endogenous ligands
14
have been reported and no validated proximal biomarkers have been described. This makes
demonstrating intracellular target engagement in living cells very challenging.
We hypothesized that we could demonstrate chemical probe 1 binding to pirin within living
cells by developing a pirinꢀtargeting Protein Degradation Probe (PDP).
RESULTS AND DISCUSSION
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Protein Degradation Probe Design. PDPs have been described against various proteins, with
16
the bromodomain epigenetic target, BRD4, the most extensively studied.
These
heterobifunctional PDP molecules have utilized several ligands that bind to the E3 ligases:
17
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VHL, the IAP proteins, and CRBN. Despite the rapid expansion in PDP research, there
remains no clear methodology to determine which proteins are amenable to PDPꢀmediated
2
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degradation, which E3 ligase ligand should be exploited or the optimal features of probe design.
The structure of the linker, its length and physicochemical properties, have all been demonstrated
21
to be important for PDP activity. The linker controls the formation of the essential ternary
22
complex, with evidence that it may stabilize the proteinꢀprotein interaction (PPI) between the
target and the E3 ligase, rather than forming a detached linear ternary complex (figure 1),
although it is unclear whether the ternary complex is part of the multiꢀprotein cullinꢀRING finger
, ,
23 24 25
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Figure 1. (Top) Pirin (5JCT)/CRBN (5CI1)/PDP ternary complex design model. The PDP can
either stabilize a PPI or simply bring the proteins in close proximity, depending on the role of the
linker. (Bottom Left) Cartoon representation of the chemical probe 1 (yellow) bound to
recombinant pirin (5JCT). The cloud represents the shape of the binding pocket with key
residues shown in black and the metal in orange. Red=oxygen, blue=nitrogen. Hydrogens and
solvent are omitted for clarity except the water coordinated to the metal, shown as a red sphere.
Both representations were generated using the PyMOL Molecular Graphics System, Version 1.8
Schrödinger, LLC. (Bottom Right) Key residues in the binding site and the clear solvent exposed
vector for the chemical probe 1 binding to pirin are shown, adapted from an analysis using MOE
2014.09. The ethyl pyrrolidine solubilizing group of chemical probe 1 was not resolved in the
crystal structure and therefore is not drawn in the analysis.
Although we had discovered a high affinity ligand for pirin, there was no evidence that this
protein would be a suitable substrate for ubiquitination by a PDPꢀrecruited E3 ligase, without
which, extensive linker optimization could be futile. Therefore, we initially designed a
synthetically tractable 15ꢀatom linker that we predicted would not affect the affinity of the PDP
2
6
for the isolated target proteins. Analysis of the crystal structure of the chemical probe 1 bound
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to pirin (figure 1, PDB: 5JCT) suggested that the solventꢀexposed solubilizing group vector
should be amenable to linker attachment. We selected a CRBNꢀtargeting thalidomide ligand as
the basis of our E3 ligase binding motif, due to its low molecular weight. The CRBNꢀtargeting
ligand would be attached via the solvent exposed hydroxyl group of the 4ꢀhydroxythalidomide
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analogue 2. The linker would then attach the pirinꢀ and CRBNꢀtargeting specific binding groups
via two amide moieties, to give our first generation pirinꢀtargeting PDP 3 (scheme 1).
Scheme 1. Design and Synthesis of the First Generation PDP 3 based on the Chemical
Probe 1
t
Reagents and conditions: (a) i) PPh , Butyl 2ꢀhydroxyacetate, DTBAD, THF, 0 ºC→RT, 16 h,
5%; ii) HCO H, DCM, 40 ºC, 16 h, 54%; iii) HATU, DIPEA, DMF, Butyl (2ꢀ(2ꢀ
aminoethoxy)ethyl)carbamate, RT, 16 h, 81%; iv) 4 M HCl in dioxane, 0.5 h, 100%; (b) i) ethyl
4ꢀbromobutanoate, K CO , DMF, RT, 16 h, 37%; (c) Herrmann’s palladacycle, Bu PHBF ,
MoCO , DBU, 2ꢀ(trimethylsilyl)ethanꢀ1ꢀol, 130 C, 62% (d) i) TBAF, THF, RT, 16 h; (e) i)
HATU, DIPEA, DMF, RT, 38% (over 2 steps); ii) LiOH.H O, MeOH/THF/H O, RT, 48 h, 18%
f) HATU, DIPEA, DMF, 74%. For the synthesis of hydroxythalidomide 2 see reference 28.
3
t
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2
2
9 t
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o
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2
2
(
The CRBNꢀtargeting motif 4 of PDP 3 was synthesized from 4ꢀhydroxythalidomide 2 in 2
28
steps and 84% yield, in a similar manner to that previously described. The pirinꢀbinding motif
of the PDP 3 was synthesized from 6ꢀbromoquinoline 5, via a palladiumꢀmediated carbonylation
reaction on the ether derivative 6. Trapping the carbonylation intermediate with 2ꢀ
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(
trimethylsilyl)ethanꢀ1ꢀol gave the silylethylꢀprotected ester 7, which facilitated TBAFꢀ
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selective hydrolysis. Amide coupling to the previously described bisꢀaniline derivative 8, was
followed by hydrolysis of the aliphatic linker ester to give acid 9. Final amide coupling to the
CRBNꢀtargeting derivative 4 gave the first generation pirinꢀtargeting PDP 3 in 10 steps and 0.4%
overall yield.
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The First Generation PDP. Analysis of the heterobifunctional PDP 3 revealed that it possessed
good affinity for recombinant pirin (table 1, entry 1) when measured using SPR, confirming the
3
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success of the rationally designed attachment vector. The affinity of PDP 3 was then assessed
against the CRBNꢀDDB1 complex, with DDB1 acting as a scaffolding protein, using an FPꢀ
31
assay similar to that previously described (figure S6). PDP 3 displayed moderate affinity for
3
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CRBNꢀDDB1, with K =230 nM, comparable to the affinities of the parent CRBN ligands,
i
thalidomide and lenalidomide (figure S7).
Following confirmation that both binding motifs of the PDP 3 retained high affinity for their
respective targets, we then investigated its activity against pirin in human cancer cell lines.
Several cell lines were assessed for CRBN expression by quantitative capillary electrophoresis
3
3
34
(
figure S17). The SKꢀOVꢀ3 ovarian carcinoma cell line displayed good basal CRBN and also
pirin expression and there was no observable depletion of pirin in these cells when treated with
chemical probe 1 (1 µM, data not shown), so this line was selected for further study.
Unfortunately, treatment of SKꢀOVꢀ3 cells with PDP 3, at high concentrations (>1 µM) and for
extended time periods (>48 h), resulted in no measurable effects on the cancer cells (data not
shown).
The Second Generation PDP. We speculated several causes for the failure of the first
generation PDP 3. Pirin may simply be incompatible with this methodology, and if so, no further
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improvements could be made. Alternatively, CRBN could be the wrong E3 ligase target to
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deplete pirin or the linker length could be inconsistent with formation of the ternary complex.
Finally, the physicochemical properties of PDP 3 could be limiting its intracellular free
3
7
concentration. Given these variables, designing a PDP against a protein target that has not
previously been validated as being susceptible to E3 ligaseꢀdirected degradation is challenging,
as optimization cycles can appear lengthy and the difficult and low yielding synthesis
discourages the generation of multiple analogues. It was also unclear how strict and narrow the
requirements for optimal PDP design would be. We hypothesized that the physicochemical
properties of the PDP were the primary cause of the failure of the first generation probe, so
began a redesign process that should increase cell membrane flux, whilst maintaining the same
linker length and CRBNꢀtargeting ligand (Table 1). By carrying out multiple changes to the PDP,
we aimed to minimize the number of iterative design cycles, so we could more rapidly validate
this approach.
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Table 1. Physicochemical Properties and Affinities for Recombinant Protein Targets of the
Three Generations of PDPs
Pirin
SPR/K
CRBN/DDB1
FP/IC50
(pIC50±SEM)
a
b
c
tPSA
(Å )
KS
(µM)
CRBN/K
i
Entry
Compd
X
R
Linker
HBD
ALogP
LogD7.4
2
d
e
D
h
f
g
(nM)
(pK
D
±SEM)
O
O
1
10 nM
850 nM
(6.07±0.14)
410 nM
(6.38±0.07)
490 nM
(6.31±0.10)
420 nM
(6.38±0.10)
1
2
3
4
3
Me
F
O
N
O
N
5
4
3
3
2.8
2.0
3.9
3.9
2.2
1.9
2.7
2.9
258
244
207
207
1
5
2
2
220
95
H
H
(6.97±0.02)
230 nM
10
16
21
(
6.63±0.01)
55 nM
7.26±0.04)
Cl
Cl
120
98
(
O
O
>
1300 nM
(<5.90)
SF=significant figure. All data was reprocessed using GraphPad Prism 7.01. See figures S8ꢀ
S16. SEM=standard error of the mean. HBD=hydrogen bond donor count. ALogP was
calculated using Biovia Pipeline Pilot Version 9.5, 2 SF. LogD measured using a HPLCꢀbased
a
b
c
7
.4
d
method, n=1, 2 SF. tPSA was calculated using ChemDraw (16.0.1.4) based on the Oꢀ and Nꢀ
e
count, 3 SF. KS=kinetic solubility in pH7.4 phosphate buffer at room temperature, n=1, 1 SF.
f
K values are reported to 2 SF and are calculated by equilibrium analysis using a one site
D
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specific binding model from SPR sensorgrams at equilibrium where possible, pK =ꢀlog (K (M)
D
D
ꢀ9
g
x 10 ) and represents the geometric mean of n=3 independent biological repeats. IC values are
5
0
reported to 2 SF and are calculated from an FPꢀassay doseꢀresponse curve to displace a
thalidomide derived fluorescent probe using a log[Inhibitor] vs. response – Variable slope (four
ꢀ
9
parameters) model, pIC =ꢀlog (IC (M) x 10 ) and represents the geometric mean of n=3
5
0
50
h
independent biological repeats, also see reference 31. K values are calculated from the
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geometric mean CRBNꢀDDB1 complex IC50 and the FPꢀprobe K using methods described in
D
reference 32.
The design of heterobifunctional molecules unavoidably results in high molecular weight
compounds, making it difficult to balance their physicochemical properties in a manner
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consistent with acceptable permeability and solubility. In the case of the first generation pirinꢀ
39
targeting PDP 3, although the LogD7.4 was acceptable (table 1, entry 1), the linker had
introduced two new hydrogen bond donors (HBD), which can have a negative impact on
2
permeability. The calculated tPSA was very high (258 Å ), although it is inevitable that tPSA
will be high for large molecules and outside the standard cutꢀoffs for cell permeability, unless the
4
0
compounds are highly lipophilic. In design a second generation PDP, we followed standard
medicinal chemistry principles to reduce the tPSA and HBD count, whilst maintaining an
acceptable LogD . To achieve this, we redesigned the ether linker in the first generation PDP 3
7
.4
to include a methylene piperazine that would project into solvent, based on analysis of the crystal
structure of the chemical probe 1 bound to pirin. The resulting tertiary amide would remove one
HBD. We also sought to mask the quinoline amide HBD, using a dipoleꢀdipole interaction, via a
41
bioisosteric replacement with fluorine. These changes resulted in the design of the second
2
generation pirinꢀtargeting PDP 10 (scheme 2) with a modestly reduced tPSA (244 Å ).
Scheme 2. Synthesis of Second (10) and Third (16) Generation Probes and Control
Compounds
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Reagents and conditions: (a) i) X=F 12, 2ꢀmethylquinoline carboxylic acid, oxalyl chloride,
DMF, DCM; RT, 3 h; then pyridine, 18 h Quant; ii) Fe(0), NH Cl, EtOH/H O, 90 ºC, 1 h, Quant;
4
2
(b) 2,3ꢀdihydrobenzoꢀ[b][1,4]ꢀdioxineꢀ6ꢀcarboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h;
then pyridine RT, 2 h, 85%; (c) i) SeO , 1,4ꢀdioxane/DMF, reflux, 1 h; ii) NꢀBocꢀpiperazine,
2
DCM, RT, 12 h then NaBH(OAc) , DCM, RT, 2 h, 94% (over 2 steps); iii) TFA, DCM, RT, 2 h,
3
t
6
9% (d) i) 2, PPh , Butyl 2ꢀhydroxyacetate, DTBAD, THF, 0 ºC→RT, 16 h, 75%; ii) HCO H,
3
2
t
DCM, 40 ºC, 16 h, 54%; iii) HATU, DIPEA, DMF, Butyl 3ꢀ(2ꢀaminoethoxy)propanoate, RT, 16
h, 72%; iv) HCO H, DCM, 40 ºC, 6 h, 93% (e) HATU, DIPEA, DMF, RT, 16 h, 52%; (f) i)
2
X=Cl 17, 2ꢀmethylquinoline carboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h; then
pyridine 2 h, 88%; ii) Fe(0), NH Cl, EtOH/H O, 90 ºC, 1 h, Quant; (g) i) 2,3ꢀdihydrobenzoꢀ
4
2
[b][1,4]ꢀdioxineꢀ6ꢀcarboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h; then pyridine 2 h,
61%; (h) i) SeO , DMF, 1,4ꢀdioxane, 50 ºC, 16 h; ii) NꢀBocꢀpiperazine, NaBH CN, AcOH,
2
3
DMF, 0 ºC→RT, 16 h iii) 4M HCl in dioxane, MeOH 0 ºC→RT, 16 h, 32% over 3 steps; (i) 2ꢀ
2ꢀ(2ꢀhydroxyethoxy)ethoxy]ethyl 4ꢀmethylbenzenesulfonate, K CO , DMF, RT, 16 h, 48%; j)
[
2
3
PPh , DTBAD, THF, RT, 2 h, 27%; (k) i) 23, 2,3ꢀdihydroꢀ1,4ꢀbenzodioxineꢀ5ꢀcarboxylic acid,
3
oxalyl chloride, DMF, DCM, RT, 2 h; then pyridine 48 h, 84%; ii) Pd/C, EtOH/DCM, H (1
2
atm), 77%; iii) 2ꢀmethylquinoline carboxylic acid, oxalyl chloride, DMF, DCM; RT, 2 h; then
pyridine 16 h, 58%; then same procedure as from 18, 6% yield over 5 steps. (l) SeO , 1,4ꢀ
2
dioxane/DMF, 50 ºC, 5.5 h; ii) Nꢀethylpiperazine, DCM, RT, 20 h; then NaBH(OAc) , DCM,
3
RT, 2 h, 35% (over 2 steps).
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The synthesis of PDP 10 began from the fluoroaniline carboxamide 11, synthesized in a
similar manner to that previously described from 2ꢀfluoroꢀ5ꢀnitroaniline 12 in 3 steps and 85%
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yield. Amide coupling to give bisamide 13 and benzylic oxidation using selenium dioxide, was
followed by reductive amination of the resulting aldehyde with NꢀBocꢀpiperazine, and
subsequent deprotection, to give 14. The CRBNꢀtargeting thalidomide derivative precursor 15
was prepared in 4 steps and 27% yield from 4ꢀhydroxythalidomide 2. Final amide coupling with
monosubstituted piperazine 14 gave the second generation pirinꢀtargeting PDP 10 in 11 steps and
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% overall yield (scheme 2).
PDP 10 displayed a similar affinity for recombinant pirin and CRBNꢀDDB1 to our first
generation probe 3 (table 1, entry 2), so was progressed to cellular assessment of pirin
degradation. Pleasingly, treatment of SKꢀOVꢀ3 ovarian cancer cells with 10 at 3.0 ꢁM total
concentration for up to 48 h revealed a clear and timeꢀdependent depletion of intracellular pirin
4
3
expression (figure S18). This confirmed, for the first time, that pirin is amenable to modulation
using a PDP and that the bisamide chemotype not only binds recombinant pirin with high
affinity, but also binds pirin within living cells.
Although demonstrating pirin depletion with the second generation probe 10 was a very
encouraging result, we found the effects were poorly reproducible. The concentrations of PDP 10
needed to observe pirin degradation were high and close to its kinetic solubility (KS).
Furthermore, at least 24 h of compound exposure was needed before pirin degradation was
observed (figure S18). To explore the cause of the variable results obtained with PDP 10, we
assessed its chemical stability. At room temperature, PDP 10 was stable as a solid and in DMSO
o
stock solution (>1 month, data not shown), but at 37 C in pH7.4 phosphate buffer, consistent
with the cell assay conditions, it underwent rapid decomposition (table S1), displaying a halfꢀlife
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of only ~4 h. This poor chemical stability was consistent with the known decomposition of the
parent CRBNꢀtargeting thalidomide ligand under these conditions, where multiple hydrolysis
44
products of the imide and glutaramide moieties are observed. The chemical probe 1 displayed
no instability under these conditions; therefore, we concluded that the facile hydrolysis of the
CRBNꢀtargeting motif limited the reproducibility of our slowꢀacting second generation pirinꢀ
targeting PDP 10.
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The Third Generation PDP. In the design of a third generation probe, we aimed to increase
permeability further. This should result in higher intracellular free concentrations more quickly,
mitigating the poor stability of the CRBNꢀtargeting motif. We decided to carry out a bioisosteric
replacement of the central ring fluorine for the larger and more sterically hindering chlorine
45
substituent. However, we were concerned that the increase in lipophilicity from this exchange
would negatively impact both the solubility and permeability of the probe. Large lipophilic
46
heterobifunctional molecules are particularly susceptible to aggregation, and this decreases the
47
free concentration that drives cell membrane flux. To balance the lipophilicity, we removed the
tertiary amide bond to the piperazine, introducing a cationic amine, which would be substantially
48
charged at pH 7.4 (Moka Version 2.5.2, pK =8.0). The second amide in the linker was also
a
removed, reducing the HBD count further and increasing the overall flexibility of the ligand,
consistent with the formation of the crucial PDP ternary protein complex. The linker length was
reduced by one atom to accommodate these changes and resulted in the design of the third
generation pirinꢀtargeting PDP 16 (CCT367766), which now displayed a notably reduced, but
2
still high, tPSA (207 Å ).
The synthesis of the pirinꢀtargeting motif of the third generation PDP 16 was carried out in a
similar manner to that previously described starting from 2ꢀchloroꢀ5ꢀnitroaniline 17, to give
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chlorobisamide 18 in 3 steps and 54% yield (scheme 2). Oxidation of the methylquinoline
moiety with SeO , reductive amination of the resulting aldehyde with NꢀBocꢀpiperazine and Nꢀ
2
Boc deprotection gave 19 in 32% yield. Following S 2 alkylation with the ether linker to give
N
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0, selective Mitsunobu alkylation with 4ꢀhydroxythalidomide 2 gave the third generation PDP
6 in 8 steps and 2% overall yield. A nonꢀpirinꢀbinding negative control matched pair
1
11
compound, PDP 21, based on our negative control pirin chemical probe, was synthesized from
4
9
regioisomer 23 in a similar manner in 2% overall yield. A nonꢀCRBN binding control 22, was
also synthesized from 18, utilizing reductive amination with Nꢀethylpiperazine, in 35% yield
(scheme 2).
Figure 2. (A) Representative SPR sensorgram of the third generation PDP 16 and recombinant
pirin. (B) Representative binding curve of PDP 16 in the CRBNꢀDDB1 FPꢀassay.
Analysis of the third generation PDP 16 confirmed that it retained acceptable lipophilicity
(table 1, entry 3), but also displayed a 4.2ꢀfold increase in affinity for recombinant pirin
compared to the second generation PDP 10 (figure 2A) and comparable affinity for CRBN
(figure 2B). SKꢀOVꢀ3 cells were then treated with PDP 16 at concentrations from 50 to 1500 nM
for up to 24 h (figure 3).
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Figure 3. A: Immunoblot of SKꢀOVꢀ3 human ovarian cancer cells demonstrating the depletion
of pirin protein using the third generation PDP 16 and the timeꢀdependent hookꢀeffect. B:
Immunoblot demonstrating the concentrationꢀdependent depletion of pirin protein after 2 h
exposure in SKꢀOVꢀ3 cells. C: Capillary electrophoresis and immunoassay were used to quantify
the pirin protein expression after 2 h exposure with PDP 16, all values are normalized to vinculin
loading control and relative to the measured basal pirin protein expression, all bars represent the
arithmetic mean of n=3 independent biological repeats, error bars are SEM. D: Proteomics
analysis of the third generation PDP 16 (50 nM) exposure (4 h) in SKꢀOVꢀ3 cells compared to
vehicle control, using a tandem mass tagging (TMT) MS2 protocol on the cell lysate, 8547
quantifiable proteins were identified, each blue dot represents a single quantifiable protein, pirin
ꢀ4
is marked in red (adjusted p value=1.4 x 10 ), p values were calculated using a linear modelling
based tꢀtest and corrected for multiple comparisons using the BenjaminiꢀHochberg method to
give the p(adj) values shown, dotted lines represent 2ꢀfold depletion of the protein and a
p(adj)=0.05.
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In contrast to our earlier generation PDPs, near complete pirin degradation was now observed
with just 50 nM treatment and only 2 h exposure (figure 3A). Increasing the total initial
concentration of pirinꢀtargeting PDP 16 resulted in a clear hookꢀeffect. This bellꢀshaped
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concentrationꢀresponse is consistent with the formation of a ternary complex. Interestingly the
hookꢀeffect was seen to decrease over time (24 h), possibly either due to the slower degradation
at high concentration or from the depletion of PDP 16 due to thalidomide hydrolysis (halfꢀ
life=~3 h, figure S1), which would reduce the effective free concentration below the negative
22
cooperativity threshold of the ternary complex. Pirin degradation was subsequently shown to be
concentrationꢀresponsive with activity at concentrations as low as 0.5 nM (figure 3B), which was
confirmed with a quantitative capillary electrophoresesꢀbased immunoassay (figure 3C). The
negative control benzodioxane regioisomer PDP 21, (table 1, entry 4) displayed no pirin
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depletion at equimolar concentrations (figure S19), and degradation was also confirmed to be
proteasomeꢀdependent by rescue following preꢀincubation with the proteosome inhibitor,
51
MG132 (500 nM, figure S19). We then carried out whole proteome mass spectrometry, to
estimate the cellular selectivity of the pirinꢀtargeting PDP 16 in an unbiased manner, quantified
using tandem mass tagging (TMT) (figure 3D, www.proteomics.com). After treating SKꢀOVꢀ3
cells with 50 nM PDP 16 for 4 h, and comparing to vehicle treated cells using Benjaminiꢀ
Hochberg corrected p values, we found that from 8547 quantifiable proteins identified, only pirin
ꢀ4
5
2
(
2.3ꢀfold reduction, p(adj)=1.4 x 10 ) displayed a statistically significant (p(adj)<0.05)
difference in protein expression.
To confirm that our chemical probe 1 also bound pirin within SKꢀOVꢀ3 cancer cells, we
carried out competition experiments, designed to rescue pirin depletion by PDP 16. The
concentrations required for a mutually exclusive binding ligand to displace a probe molecule, are
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dependent on the affinity of the ligand relative to the ratio of the free concentration of the probe
53
and its affinity for the protein target complex. Because the depletion of pirin is a nonꢀ
equilibrium event that does not necessarily require complete target occupancy, it is more difficult
1
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to observe competition at later time points owing to continued protein turnover. Pretreating SKꢀ
OVꢀ3 cells with 10 µM thalidomide, as a CRBNꢀbinding competitive ligand, demonstrated that
after 2 h treatment with PDP 16 (5 nM) we successfully rescued pirin depletion (figure S19). The
nonꢀCRBNꢀbinding control chlorobisamide 22, displayed high affinity for recombinant pirin
(
SPR, K =21 nM, pK =7.68±0.03, n=3). Pretreatment of SKꢀOVꢀ3 cells with chlorobisamide 22
D D
displayed concentrationꢀdependent rescue of pirin expression, following treatment with the PDP
6 (figure 4, top), with complete rescue observed at 1 µM. Finally, preꢀtreatment with the
1
chemical probe 1 demonstrated clear rescue of pirin depletion, confirming intracellular target
engagement (figure 4, bottom).
Figure 4. Intracellular competition studies with PDP 16 and the chemical probes. SKꢀOVꢀ3 cells
were preꢀtreated with increasing concentrations of chemical probe for 4 h before exposing to
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PDP 16 for 2 h at the concentrations shown. Cells were then lysed and protein expression
analyzed using immunoblot. For clarity, gel images have been cropped where appropriate.
CONCLUSION
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Exploiting cellꢀbased phenotypic screens to identify new diseaseꢀassociated therapeutic targets
is an increasingly frequent strategy in drug discovery. While this approach can identify novel
targets with unique mechanisms of action, these proteins are often poorly characterized and can
lack identifiable enzymatic activity, ligands and biomarkers of target engagement. The main
focus of research into PDPs has been as potential therapeutics with novel mechanisms of action.
We designed a PDP as an intracellular probe against the poorlyꢀunderstood and nonꢀcatalytic
molecular target, pirin. Developing PDPs to confirm intracellular target engagement, and
potentially develop intracellular SAR, against challenging proteins, is an important addition to
the current methods for compound profiling.
For PDPs to be used as target engagement probes, their rapid development and validation is
crucial. The ideal strategies for efficient and successful PDP design are still under investigation
and will clearly improve as more protein targets are modulated and additional crystallographic
evidence of the target protein/E3 ligase/PDP ternary complexes are discovered. The number of
variables involved in PDP design against nonꢀvalidated target proteins can make the process
daunting. By focusing on the physicochemical properties of our probe molecules, in only three
iterations, we developed a selective degradation probe that eliminates pirin at low concentration
and in a short timeꢀperiod. This confirmed our chemical probe 1 does bind pirin in an
intracellular environment and PDP 16 provides another chemical tool to study a largely
unexplored protein.
EXPERIMENTAL SECTION
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General Experimental.
Unless otherwise stated, reactions were conducted in oven dried glassware under an
atmosphere of nitrogen or argon using anhydrous solvents. All commercially obtained reagents
and solvents were used as received. Thin layer chromatography (TLC) was performed on preꢀ
coated aluminum sheets of silica (60 F254 nm, Merck) and visualized using shortꢀwave UV
light. Flash column chromatography was carried out on Merck silica gel 60 (partial size 40ꢀ65
1
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ꢁ
m). Column chromatography was also performed on a Biotage SP1 or Biotage Isolera Four
purification system using Biotage Flash silica cartridges (SNAP KPꢀSil) or for reverse phase
purifications SNAP Ultra C18 cartridges. Ion exchange chromatography was performed using
acidic Isolute Flash SCXꢀII columns. Semiꢀpreparative HPLC was performed on an Agilent 6120
system, flow 20 mL/min, eluents 0.1% acetic acid in water and 0.1% acetic acid in methanol,
gradient of 10% to 100% organic phase. Lipophilic method: Chromatographic separation at room
temperature was carried out using a 1200 Series Preparative HPLC (Agilent, Santa Clara, USA)
over a 15 minute gradient elution (Grad15min,20mls) from 60:40 to 0:100 water:methanol (both
1
modified with 0.1% formic acid) at a flow rate of 20 mL/min. HꢀNMR spectra were recorded on
Bruker AMX500 (500 MHz) spectrometers using an internal deuterium lock. Chemical shifts are
quoted in parts per million (ppm) using the following internal references: CDCl (δH 7.26),
3
MeOD (δH 3.31) and DMSOꢀd (δH 2.50). Signal multiplicities are recorded as singlet (s),
6
doublet (d), triplet (t), quartet (q) and multiplet (m), doublet of doublets (dd), doublet of doublet
of doublets (ddd), broad (br) or obscured (obs). Coupling constants, J, are measured to the
13
nearest 0.1 Hz. CꢀNMR spectra were recorded on Bruker AMX500 spectrometers at 126 MHz
using an internal deuterium lock. Chemical shifts are quoted to 0.01 ppm, unless greater
accuracy was required, using the following internal references: CDCl (δC 77.0), MeOD (δC
3
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9.0) and DMSOꢀd (δC 39.5). High resolution mass spectra were recorded on an Agilent 1200
6
series HPLC and diode array detector coupled to a 6210 time of flight mass spectrometer with
dual multimode APCI/ESI source or on a Waters Acquity UPLC and diode array detector
coupled to a Waters G2 QToF mass spectrometer fitted with a multimode ESI/APCI source. All
compounds were >95% purity by LCMS analysis unless otherwise stated.
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Thalidomide (http://www.sigmaaldrich.com/catalog/product/sigma/t144?lang=en&region=GB,
Accessed
http://www.sigmaaldrich.com/catalog/product/aldrich/cds022536?lang=en&region=GB,
Accessed August 29, 2017) and
(http://www.sigmaaldrich.com/catalog/product/sigma/m8699?lang=en&region=GB,
August
29,
2017),
Lenalidomide
(
MG132
Accessed
August 29, 2017) were purchased from Sigma Aldrich and used without further purification.
The pirin chemical probe 1 CCT251236 was synthesized using the previously described
1
1
procedure.
2
ꢀ(2,6ꢀDioxopiperidinꢀ3ꢀyl)ꢀ4ꢀhydroxyisoindolineꢀ1,3ꢀdione 2
3
ꢀAminopiperidineꢀ2,6ꢀdione hydrochloride (300 mg, 1.82 mmol) was added to a solution of
4
ꢀhydroxyisobenzofuranꢀ1,3ꢀdione (299 mg, 1.82 mmol) and triethylamine (0.38 mL, 2.73
mmol) in anhydrous THF (36.5 mL) and the reaction heated to reflux for 24 h under inert
atmosphere. The reaction mixture was allowed to cool to room temperature, then 1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide (384 mg, 2.00 mmol) and DMAP (22 mg, 0.18 mmol) were
added. The reaction was heated to reflux for 24 h. Then, the reaction mixture was allowed to cool
and the solvents were removed under reduced pressure. The resulting residue was taken up in
methanol and passed sequentially through two ion exchange columns (Isolute SCXꢀII), eluting
1
with methanol to afford the title compound as an amorphous offꢀwhite solid (422 mg, 84%). H
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NMR (500 MHz, DMSOꢀd ) δ 11.18 (s, 1H), 11.09 (s, 1H), 7.65 (dd, J = 8.4, 7.2 Hz, 1H), 7.32
6
(dd, J = 7.1, 0.7 Hz, 1H), 7.25 (dd, J = 8.4, 0.7 Hz, 1H), 5.07 (dd, J = 12.8, 5.5 Hz, 1H), 2.88
(ddd, J = 17.0, 13.9, 5.5 Hz, 1H), 2.64 – 2.46 (m, 2H), 2.02 (dtd, J = 13.0, 5.3, 2.2 Hz, 1H).
+
+
+
28
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
LCMS (ESI ) RT = 0.77 min, 100%, M+H 275, M+Na 297. This compound is also
commercially available from several suppliers.
tertꢀButyl 2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetate
Triphenylphosphine (0.261 g, 0.996 mmol) and tertꢀbutyl 2ꢀhydroxyacetate (101 mg, 0.77
mmol) were dissolved in anhydrous THF (3 mL), while stirring at 0 ºC. Then, a solution of
DTBAD (229 mg, 0.10 mmol) in anhydrous THF (2 mL), was added dropwise. Finally, a
solution of (2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ4ꢀhydroxyisoindolineꢀ1,3ꢀdione) 2 in anhydrous THF (3
mL) was added. The mixture was stirred for 1 h at 0 ºC, then allowed to warm to room
temperature and stirred overnight. Then, the solvent was removed under reduced pressure. The
crude product was purified by Biotage chromatography using a gradient of 0 to 50% EtOAc in
cyclohexane, to afford the title compound as an amorphous white solid (224 mg, 0.577 mmol, 75
1
%
yield). H NMR (500 MHz, DMSOꢀd ) δ 11.11 (s, 1H), 7.80 (dd, J = 8.5, 7.3 Hz, 1H), 7.48 (d,
6
J = 7.2 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 5.10 (dd, J = 12.8, 5.5 Hz, 1H), 4.97 (s, 2H), 2.89 (ddd,
13
J = 17.0, 13.9, 5.4 Hz, 1H), 2.64 – 2.52 (m, 2H), 2.07 – 2.00 (m, 1H), 1.43 (s, 9H). C NMR
(
126 MHz, DMSOꢀd ) δ 172.78, 169.89, 167.14, 166.71, 165.12, 155.03, 136.76, 133.25, 119.96,
6
+
116.44, 115.90, 81.92, 65.50, 48.80, 30.95, 27.68, 21.97. HRMS (ESI ): calcd for C H N O
1
5
13
2
7
t
+
(
M + H, ꢀ Bu) 333.0717, found 333.0722.
2ꢀ((2ꢀ(2,6ꢀDioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetic acid
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
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5
5
5
5
5
5
5
6
tertꢀButyl 2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetate (1.30 g, 3.35
mmol) was dissolved in anhydrous DCM, then formic acid (12.8 mL, 335 mmol), was added
while stirring at room temperature. The reaction was stirred overnight at 40 ºC. The solvents
were removed under reduced pressure and the resulting residue purified by reverse phase Biotage
chromatography using a gradient of 0% to 50% MeOH in water + 0.1% formic acid, to afford the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
title compound as an amorphous white solid (600 mg, 1.81 mmol, 54% yield). H NMR (500
MHz, DMSOꢀd ) δ 13.29 (s, 1H), 11.11 (s, 1H), 7.79 (dd, J = 8.5, 7.3 Hz, 1H), 7.47 (d, J = 7.2
6
Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 5.10 (dd, J = 12.8, 5.4 Hz, 1H), 4.98 (s, 2H), 2.89 (ddd, J =
+
1
6.9, 13.9, 5.4 Hz, 1H), 2.64 – 2.52 (m, 2H), 2.04 (dtd, J = 13.0, 5.4, 2.3 Hz, 1H). LCMS (ESI ):
+
28
RT = 0.71 min, 100%, M+Na 355.
tertꢀButyl
3ꢀ(2ꢀ(2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀ
yl)oxy)acetamido)ethoxy)propanoate
ꢀ((2ꢀ(2,6ꢀDioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetic acid (217 mg, 0.65
mmol), tertꢀbutyl 3ꢀ(2ꢀaminoethoxy)propanoate (124 mg, 0.65 mmol) and DIPEA (0.34 mL,
.96 mmol) were dissolved in anhydrous DMF (3.27 mL) at room temperature under inert
2
1
atmosphere. HATU (230 mg, 0.98 mmol) was added and the reaction stirred overnight. The
solvents were removed under reduced pressure and the resulting residue was purified by reverse
phase Biotage chromatography using a gradient of 10ꢀ90% MeOH in water + 0.1% formic acid
1
to afford the title compound as an amorphous white solid (236 mg, 72%). H NMR (500 MHz,
MeOD) δ 7.82 (dd, J = 8.4, 7.4 Hz, 1H), 7.55 (dd, J = 7.3, 0.5 Hz, 1H), 7.46 – 7.43 (m, 1H), 5.16
(dd, J = 12.6, 5.5 Hz, 1H), 4.78 (s, 2H), 3.71 (t, J = 6.2 Hz, 2H), 3.62 – 3.56 (m, 2H), 3.52 – 3.48
(m, 2H), 2.90 (ddd, J = 18.0, 14.4, 5.2 Hz, 1H), 2.82 – 2.70 (m, 2H), 2.50 (td, J = 6.2, 3.8 Hz,
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13
2
H), 2.16 (dddd, J = 10.8, 7.9, 5.1, 2.4 Hz, 1H), 1.42 (s, 9H). C NMR (126 MHz, MeOD) δ
1
74.55, 172.81, 171.33, 170.00, 168.31, 167.59, 156.21, 138.16, 134.96, 121.60, 119.30, 117.89,
+
81.75, 70.01, 69.26, 67.75, 50.56, 40.15, 37.22, 32.20, 28.34, 23.66. HRMS (ESI ): calcd for
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C H N O (M + H) 504.1982, found 504.1981.
24
30
3
9
tertꢀButyl
yl)oxy)acetamido)ethoxy)ethyl)carbamate
ꢀ((2ꢀ(2,6ꢀDioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetic acid (0.19 g, 0.56 mmol),
(2ꢀ(2ꢀ(2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀ
2
tertꢀbutyl (2ꢀ(2ꢀaminoethoxy)ethyl)carbamate (0.11 g, 0.56 mmol) and DIPEA (0.29 ml, 1.68
mmol) were dissolved in anhydrous DMF (2.80 mL) under inert atmosphere at room
temperature. HATU (198 mg, 0.84 mmol) was added and the reaction stirred for 16 h. The
reaction mixture was concentrated in vacuo and the resulting residue purified by Biotage
chromatography using a gradient of 0ꢀ2% methanol in ethyl acetate. However, this material was
not pure, therefore the material was purified by further Biotage chromatography using a gradient
of 1ꢀ10% EtOH in DCM to afford the title compound as an amorphous white solid (243 mg,
1
8
1%). H NMR (500 MHz, DMSOꢀd ) δ 11.11 (s, 1H), 8.02 (t, J = 5.4 Hz, 1H), 7.81 (dd, J = 8.5,
6
7
.3 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 6.76 (t, J = 4.7 Hz, 1H), 5.12 (dd,
J = 12.8, 5.4 Hz, 1H), 4.79 (s, 2H), 3.44 (t, J = 5.6 Hz, 2H), 3.38 (t, J = 6.1 Hz, 2H), 3.31 (d, J =
.5 Hz, 2H), 3.08 (q, J = 5.6 Hz, 2H), 2.89 (ddd, J = 16.6, 13.8, 5.3 Hz, 1H), 2.65 – 2.51 (m,
5
1
3
2H), 2.08 – 1.98 (m, 1H), 1.36 (s, 9H). C NMR (126 MHz, DMSOꢀd ) δ 172.73, 169.85,
6
1
6
66.88, 166.80, 166.72, 165.45, 155.59, 155.00, 136.92, 133.03, 120.34, 116.75, 116.02, 77.63,
+
9.02, 68.55, 67.48, 48.80, 38.38, 30.95, 28.22, 21.99. HRMS (ESI ): calcd for C H N O Na
2
4
30
4
9
+
(M + Na) 541.1911, found 541.1915.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Nꢀ(5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀmethylphenyl)ꢀ2ꢀ(4ꢀ((2ꢀ(2ꢀ(2ꢀ((2ꢀ
(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetamido)ethoxy)ethyl)amino)ꢀ4ꢀ
oxobutoxy)quinolineꢀ6ꢀcarboxamide 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Nꢀ(2ꢀ(2ꢀAminoethoxy)ethyl)ꢀ2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀ
yl)oxy)acetamide
hydrochloride
4
(21.0
mg,
0.046
mmol),
4ꢀ((6ꢀ((5ꢀ(2,3ꢀ
dihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀmethylphenyl)carbamoyl)quinolinꢀ2ꢀ
yl)oxy)butanoic acid (25.0 mg, 0.046 mmol) and DIPEA (0.032 mL, 0.184 mmol) were
dissolved in anhydrous DMF (0.23 mL) under inert atmosphere. HATU (16.0 mg, 0.069 mmol)
was added and the reaction left to stir at room temperature for 2 h. The reaction was concentrated
in vacuo, then purified by reverse phase Biotage chromatography using a gradient of 20ꢀ100%
MeOH in water + 0.1% formic acid to afford the title compound as a beige solid (32 mg, 74%).
1
H NMR (500 MHz, DMSOꢀd ) δ 11.09 (s, 1H), 10.07 (s, 1H), 10.06 (s, 1H), 8.56 (d, J = 1.9 Hz,
6
1H), 8.37 (d, J = 8.8 Hz, 1H), 8.21 (dd, J = 8.8, 2.1 Hz, 1H), 8.01 (t, J = 5.5 Hz, 1H), 7.91 (t, J =
5.5 Hz, 1H), 7.87 – 7.83 (m, 2H), 7.80 (dd, J = 8.5, 7.3 Hz, 1H), 7.58 (dd, J = 8.3, 2.1 Hz, 1H),
7.54 (d, J = 2.1 Hz, 1H), 7.51 (dd, J = 8.4, 2.2 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 8.5
Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 5.12 (dd, J =
1
2.8, 5.4 Hz, 1H), 4.79 (s, 2H), 4.42 (t, J = 6.5 Hz, 2H), 4.33 – 4.26 (m, 4H), 3.45 (t, J = 5.7 Hz,
H), 3.42 (t, J = 5.9 Hz, 2H), 3.36 – 3.28 (m, J = 5.5 Hz, 2H – obscured by water peak), 3.24 (q,
2
J = 5.8 Hz, 2H), 2.89 (ddd, J = 16.3, 13.6, 5.1 Hz, 1H), 2.64 – 2.52 (m, 2H), 2.29 (t, J = 7.3 Hz,
13
2
H), 2.23 (s, 3H), 2.11 – 1.93 (m, 3H). C NMR (126 MHz, DMSOꢀd ) δ 172.74, 171.66,
6
1
69.87, 166.90, 166.71, 165.44, 164.91, 164.34, 162.79, 154.96, 147.58, 146.33, 142.92, 140.16,
137.28, 136.90, 136.33, 133.01, 130.10, 130.00, 128.75, 128.37, 128.10, 127.68, 126.72, 123.99,
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1
6
21.19, 120.32, 118.61, 118.11, 116.83, 116.74, 116.65, 116.02, 113.94, 68.91, 68.58, 67.48,
5.38, 64.39, 64.02, 48.80, 38.44, 38.33, 31.78, 30.94, 24.62, 21.99, 17.48. HRMS: calcd for
+
C H N O (M+H) , 942.3305; found 942.3322.
49
48
7
13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Nꢀ(2ꢀ(2ꢀAminoethoxy)ethyl)ꢀ2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀ
yl)oxy)acetamide 4
tertꢀButyl
(2ꢀ(2ꢀ(2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀ
yl)oxy)acetamido)ethoxy)ethyl)carbamate (24 mg, 0.05 mmol) was stirred in 4 M HCl in
dioxane (1.16 mL, 4.63 mmol) at room temperature. After 30 min, the solution was concentrated
+
and used as the hydrochloride salt without further purification. LCMS (ESI ): RT = 0.63 min,
+
1
00%, M+H 419.
Ethyl 4ꢀ((6ꢀbromoquinolinꢀ2ꢀyl)oxy)butanoate 6
6
ꢀBromoquinolinꢀ2(1H)ꢀone 5 (200 mg, 0.89 mmol) was dissolved in anhydrous DMF (4.5
mL) at room temperature under nitrogen and potassium carbonate (185 mg, 1.34 mmol) was
added. The reaction mixture was allowed to stir for 1.5 h, then ethyl 4ꢀbromobutanoate (0.26 mL,
1
.79 mmol) was added dropwise and the reaction stirred overnight. The reaction mixture was
poured into water (50 ml) and the aqueous layer extracted with DCM (3 x 15 ml). The combined
organic layer was dried (Na SO ) and concentrated in vacuo to afford the crude product as an
2
4
orange oil. This material was purified by Biotage chromatography using a gradient of 0ꢀ100%
EtOAc in cyclohexane to afford the required Oꢀlinked isomer as a white solid (111 mg, 37%)
and the undesired Nꢀlinked isomer as a yellow oil (164 mg, 55%). The regioisomers were
1
distinguishable by NOE spectroscopy. H NMR (500 MHz, CDCl ) δ 7.91 (d, J = 8.9 Hz, 1H),
3
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.88 – 7.87 (br m, 1H), 7.71– 7.69 (m, 2H), 6.92 (d, J = 8.8 Hz, 1H), 4.52 (t, J = 6.3 Hz, 2H),
.18 (q, J = 7.1 Hz, 2H), 2.55 (t, J = 7.5 Hz, 2H), 2.19 (ddd, J = 13.7, 7.4, 6.3 Hz, 2H), 1.28 (t, J
4
1
3
= 7.1 Hz, 3H). C NMR (126 MHz, CDCl ) δ 173.39, 162.35, 145.41, 137.80, 132.82, 129.61,
3
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
29.11, 126.42, 117.29, 114.30, 65.17, 60.59, 31.27, 24.60, 14.39. HRMS (ESI ): calcd for
79
+
C H BrNO (M+H) , 339.0419; found 339.0403.
15
17
3
2
ꢀ(Trimethylsilyl)ethyl 2ꢀ(4ꢀethoxyꢀ4ꢀoxobutoxy)quinolineꢀ6ꢀcarboxylate 7
Ethyl 4ꢀ((6ꢀbromoquinolinꢀ2ꢀyl)oxy)butanoate (554 mg, 1.64 mmol), tri-tertꢀ
6
butylphosphonium tetrafluoroborate (95 mg, 0.33 mmol) and Herrmann’s palladacyle (77 mg,
0.08 mmol) were added to a microwave vial and suspended in 2ꢀ(trimethylsilyl)ethanol (15 ml).
Molybdenum hexacarbonyl (865 mg, 3.28 mmol) followed by DBU 1.0 M in THF (4.91 mL,
4
.91 mmol) were then added and the vial promptly sealed. The reaction was heated to 130 °C for
1 h in a microwave. The reaction mixture was diluted with DCM and filtered to remove solids.
The filtrate was concentrated in vacuo. This residue was diluted with water (20 ml) and extracted
with DCM (3 x 20 ml). The combined organic layer was washed with brine and concentrated
under reduced pressure. The resulting crude was purified by Biotage chromatography using a
gradient of 0ꢀ100% EtOAc in cyclohexane to give 472 mg product (88% purity by LCMS, 62%),
1
as a brown oil. H NMR (500 MHz, CDCl ) δ 8.47 (d, J = 1.9 Hz, 1H), 8.23 (dd, J = 8.7, 1.9 Hz,
3
1
H), 8.06 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 4.56 (t, J = 6.3
Hz, 2H), 4.51 – 4.43 (m, 2H), 4.17 (q, J = 7.1 Hz, 2H), 2.55 (t, J = 7.4 Hz, 2H), 2.19 (p, J = 7.0,
+
6
.5 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H), 1.23 – 1.15 (m, 2H), 0.12 (s, 9H). LCMS (ESI ): RT =1.79
+
min, 88%, (M+H) 404.
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Ethyl
4ꢀ((6ꢀ((5ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀ
methylphenyl)carbamoyl)quinolinꢀ2ꢀyl)oxy)butanoate.
To a stirring solution of 2ꢀ(trimethylsilyl)ethyl 2ꢀ(4ꢀethoxyꢀ4ꢀoxobutoxy)quinolineꢀ6ꢀ
carboxylate 7 (463 mg, 1.15 mmol) in anhydrous THF (12 mL) at room temperature under
nitrogen was dropwise added TBAF 1.0 M in THF (1.72 mL, 1.72 mmol). The reaction was
allowed to stir at RT overnight, after which time the starting material had been consumed as
indicated by LCMS. The reaction mixture was diluted with water and concentrated in vacuo to
remove the THF. The aqueous layer was acidified to ~pH 2 with 1M HCl aq. and extracted with
DCM (3 x 15 ml). The combined organic layer was washed with brine (20 mL) and dried
(Na SO ) to afford the crude product as a yellow oil. This material was used directly in the next
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
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4
4
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4
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5
5
5
5
5
5
5
6
0
1
2
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4
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6
7
8
9
0
1
2
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4
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7
8
9
0
1
2
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9
0
1
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
+
+
step without further purification. LCMS (ESI ): RT = 1.57 min, 84%, (M+H) 304.
ꢀ(4ꢀEthoxyꢀ4ꢀoxobutoxy)quinolineꢀ6ꢀcarboxylic acid (74 mg, 0.24 mmol) was dissolved in
2
anhydrous DMF (2 ml) and DIPEA (0.12 ml, 0.67 mmol) was added, followed by HATU (105
mg, 0.28 mmol). The reaction was allowed to stir for 5 min and then Nꢀ(3ꢀaminoꢀ4ꢀ
methylphenyl)ꢀ2,3ꢀdihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamide (63 mg, 0.22 mmol) was added.
The reaction was stirred at room temperature overnight. The reaction mixture was poured into
water and the resulting precipitate collected by filtration. The precipitate was dissolved in
DCM/MeOH and preꢀabsorbed onto silica. The crude material was purified by Biotage
chromatography using a gradient of 0ꢀ5% MeOH in DCM and then by preparative HPLC (20
mls, lipophilic method) to afford the title compound as an amorphous beige solid (48 mg, 38%
1
over two steps). H NMR (500 MHz, CDCl ) δ 8.30 (d, J = 1.9 Hz, 1H), 8.13 – 8.05 (m, 3H),
3
7
.93 – 7.87 (m, 3H),7.68 (dd, J = 8.3, 2.1 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.38 (dd, J = 8.4, 2.2
Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.57 (t, J =
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2
2
2
2
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3
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.3 Hz, 2H), 4.31 (ddd, J = 11.1, 3.7, 1.8 Hz, 4H), 4.18 (q, J = 7.1 Hz, 2H), 2.56 (t, J = 7.4 Hz,
1
3
2
H), 2.35 (s, 3H), 2.21 (p, J = 6.7 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, CDCl )
3
δ 173.37, 165.49, 165.09, 163.48, 148.69, 146.89, 143.67, 139.54, 136.92, 136.11, 131.17,
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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8
9
0
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9
0
1
1
30.22, 128.23, 128.05, 127.62, 127.30, 125.17, 124.61, 120.59, 117.63, 117.52, 116.89, 114.86,
+
14.58, 65.40, 64.71, 64.34, 60.62, 31.26, 24.58, 17.52, 14.39. HRMS (ESI ) : calcd for
+
C H N O (M+H) , 570.2253; found 570.2222.
32 32
3
7
4
ꢀ((6ꢀ((5ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀ
methylphenyl)carbamoyl)quinolinꢀ2ꢀyl)oxy)butanoic acid 9.
To a stirring solution of ethyl 4ꢀ((6ꢀ((5ꢀ(2,3ꢀdihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀ
methylphenyl)carbamoyl)quinolinꢀ2ꢀyl)oxy)butanoate (37 mg, 0.065 mmol) in THF (0.5 mL)
and MeOH (0.25 mL) was added a solution of LiOH.H O (13.6 mg, 0.325 mmol) in water (0.25
2
mL). After 2 days the reaction mixture was diluted with water (30 ml) and the aqueous layer
acidified to ~pH 2 with 1M HCl aq. The aqueous layer was extracted with DCM (3 x 15 ml). The
combined organic layer was washed with 1M HCl aq. (20 ml), brine (20 ml) and dried (Na SO ),
2
4
then concentrated in vacuo to afford the product as an amorphous pale yellow solid (6.5 mg,
1
1
8%). This material was used directly in the next reaction without further purification. H NMR
(500 MHz, DMSOꢀd ) δ 12.16 (br s, 1H), 10.07 (s, 2H), 8.56 (d, J = 1.9 Hz, 1H), 8.38 (d, J = 8.8
6
Hz, 1H), 8.22 (dd, J = 8.7, 2.1 Hz, 1H), 7.89 – 7.83 (m, 2H), 7.58 (dd, J = 8.3, 2.1 Hz, 1H), 7.54
(d, J = 2.1 Hz, 1H), 7.51 (dd, J = 8.4, 2.2 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.11 (d, J = 8.8 Hz,
1
3
H), 6.99 (s, 1H), 4.47 (t, J = 6.5 Hz, 2H), 4.34 – 4.21 (m, 4H), 2.43 (t, J = 7.4 Hz, 2H), 2.24 (s,
1
3
H), 2.03 (p, J = 7.0 Hz, 2H). C NMR (126 MHz, DMSO-d ) δ 174.11, 164.94, 164.35, 162.78,
6
147.57, 146.34, 142.93, 140.23, 137.29, 136.33, 130.11, 130.05, 128.76, 128.40, 128.12, 127.68,
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26.75, 124.03, 121.20, 118.61, 118.11, 116.84, 116.66, 113.94, 65.06, 64.39, 64.02, 30.30,
+
+
4.05, 17.49. HRMS (ESI ): calcd for C H N O (M+H) , 542.1922; found 542.1905.
3
0
28
3
7
1
1
1
1
1
1
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0
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9
0
Nꢀ(2ꢀFluoroꢀ5ꢀnitrophenyl)ꢀ2ꢀmethylquinolineꢀ6ꢀcarboxamide
Oxalyl chloride (3.25 mL, 38.4 mmol) was added dropwise to a solution of 2ꢀmethylquinolineꢀ
ꢀcarboxylic acid (6.59 g, 35.2 mmol) and DMF (0.0062 mL, 0.080 mmol) in anhydrous DCM
6
(80 mL). The reaction mixture was stirred at room temperature for 3 h and then concentrated
under reduced pressure. The residue was dissolved in DCM (30 mL) and concentrated again
under reduced pressure. The resulting dry residue was dissolved in pyridine (80 mL) and 2ꢀ
fluoroꢀ5ꢀnitroaniline 12 (5.00 g, 32.0 mmol) was added in one portion. The reaction mixture was
stirred at room temperature for 18 h and then poured onto water (100 mL). The green precipitate
was filtered and washed with water (3 x 20 mL), diethyl ether (3 x 20 mL) and DCM (10 mL), to
afford the title compound as a light green solid, which was carried onto the next step without
1
further purification (10.4 g, Quant.). H NMR (500 MHz, DMSOꢀd ) δ 10.71 (s, 1H), 8.72 (dd, J
6
=
6.5, 2.9 Hz, 1H), 8.63 (d, J = 2.0 Hz, 1H), 8.43 (d, J = 8.5 Hz, 1H), 8.23 (dd, J = 8.8, 2.0 Hz,
1
H), 8.21 – 8.16 (m, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.65 (app t, J = 9.5 Hz, 1H), 7.55 (d, J = 8.4
13
Hz, 1H), 2.71 (s, 3H). C NMR (126 MHz, DMSOꢀd ) δ 165.53, 161.22, 158.67 (d, J = 258.2
6
Hz), 148.65, 143.72, 137.32, 130.36, 128.88, 128.48, 128.00, 127.08 (d, J = 13.9 Hz), 125.33,
1
9
1
23.18, 122.14 (d, J = 9.6 Hz), 121.25 (d, J = 3.8 Hz), 117.19 (d, J = 22.8 Hz), 25.07. F NMR
+
+
(470 MHz, DMSOꢀd ) δ ꢀ110.20. HRMS (ESI ): calcd for C H FN O (M + H) , 326.0935;
6
17 13
3
3
found 326.0931.
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Nꢀ(5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀfluorophenyl)ꢀ2ꢀ((4ꢀ(3ꢀ(2ꢀ(2ꢀ((2ꢀ
(
2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀyl)oxy)acetamido)ethoxy)propanoyl)piperazinꢀ
1ꢀyl)methyl)quinolineꢀ6ꢀcarboxamide 10
ꢀ(2ꢀ(2ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1,3ꢀdioxoisoindolinꢀ4ꢀ
0
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0
3
yl)oxy)acetamido)ethoxy)propanoic acid 15 (15 mg, 0.033 mmol),
Dihydrobenzo[b][1,4]dioxineꢀ6ꢀcarboxamido)ꢀ2ꢀfluorophenyl)ꢀ2ꢀ(piperazinꢀ1ꢀ
N
Nꢀ(5ꢀ(2,3ꢀ
ylmethyl)quinolineꢀ6ꢀcarboxamide 14 (18 mg, 0.033 mmol) and HATU (19 mg, 0.050 mmol)
were dissolved in anhydrous DMF (0.34 ml) at room temperature under inert atmosphere.
DIPEA (23.4 ꢁL, 0.134 mmol) was added and the reaction stirred for 16 h. The reaction mixture
was concentrated in vacuo and purified by reverse phase Biotage chromatography (10ꢀ80%
MeOH in water + 0.1% formic acid), however the material obtained was not pure. Further
purification by Biotage chromatography using a gradient of 2ꢀ50% EtOH in DCM afforded the
1
product as an offꢀwhite amorphous solid (17 mg, 52%). H NMR (500 MHz, DMSOꢀd ) δ 11.12
6
(
s, 1H), 10.39 (s, 1H), 10.18 (s, 1H), 8.65 (d, J = 2.0 Hz, 1H), 8.50 (d, J = 8.4 Hz, 1H), 8.25 (dd,
J = 8.8, 2.0 Hz, 1H), 8.14 (dd, J = 7.1, 2.6 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.99 (t, J = 5.6 Hz,
1
H), 7.80 (dd, J = 8.5, 7.3 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.65 (ddd, J = 9.0, 4.4, 2.7 Hz, 1H),
.56 – 7.46 (m, 3H), 7.39 (d, J = 8.5 Hz, 1H), 7.30 (dd, J = 10.1, 9.0 Hz, 1H), 6.99 (d, J = 8.4
7
Hz, 1H), 5.12 (dd, J = 12.8, 5.4 Hz, 1H), 4.79 (s, 2H), 4.31 (td, J = 5.3, 3.6 Hz, 4H), 3.82 (s, 2H),
.62 (t, J = 6.6 Hz, 2H), 3.52 – 3.41 (m, 6H), 3.33 (s, 2H), 2.89 (ddd, J = 16.7, 13.7, 5.4 Hz, 1H),
3
13
2.62 – 2.52 (m, 4H), 2.43 (d, J = 21.7 Hz, 4H), 2.09 – 1.99 (m, 1H). C NMR (126 MHz,CDCl )
3
δ 171.71, 169.89, 168.79, 167.12, 166.76, 166.06, 165.15, 165.08, 161.23, 154.62, 149.43 (d, J =
2
40.9 Hz), 149.10, 146.95, 143.59, 137.71, 137.06, 134.97, 133.71, 131.94, 129.99, 127.99,
127.94 (d, J = 9.5 Hz), 127.28, 126.81, 126.42 (d, J = 11.2 Hz), 122.32, 120.74, 119.68, 118.21,
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17.48, 117.46 (d, J = 6.5 Hz), 117.05 (d, J = 7.0 Hz), 116.93, 115.30 (d, J = 20.0 Hz), 113.74,
9.20, 68.22, 67.52, 64.74, 64.70, 64.32, 53.39, 53.15, 49.42, 45.80, 41.68, 39.47, 33.60, 31.52,
+
+
23.00. HRMS (ESI ): calcd for C H FN O (M + H) , 971.3370; found 971.3343.
5
0
48
8
12
1
1
1
1
1
1
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1
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9
0
Nꢀ(5ꢀAminoꢀ2ꢀfluorophenyl)ꢀ2ꢀmethylquinolineꢀ6ꢀcarboxamide 11
To a solution of Nꢀ(2ꢀfluoroꢀ5ꢀnitrophenyl)ꢀ2ꢀmethylquinolineꢀ6ꢀcarboxamide (10.4 g, 32.0
mmol) in ethanol (120 mL) and water (40 mL), ammonium chloride (12.0 g, 224 mmol) and iron
powder (12.5 g, 224 mmol) were added in one portion and the resulting suspension was allowed
to stir at 90 °C for 1 h. The reaction mixture was allowed to cool to room temperature, diluted
with MeOH (20 mL) and DCM (20 mL) and filtered through a pad of Celite. The resulting
filtrate was concentrated under vacuum to afford a light brown solid which was reꢀdissolved in a
mixture of DCM:MeOH (9:1, 150 mL) and washed with saturated aqueous NaHCO (150 mL).
3
The organic phase was dried over Na SO , filtered and concentrated under reduced pressure to
2
4
afford a yellow solid as crude product, which was taken directly onto the next step without
1
further purification (9.46 g, Quant.). H NMR (500 MHz, DMSOꢀd ) δ 10.05 (s, 1H), 8.57 (d, J =
6
1
7
6
1
.8 Hz, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.19 (dd, J = 8.8, 2.0 Hz, 1H), 8.01 (d, J = 8.8 Hz, 1H),
.52 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 10.3, 8.8 Hz, 1H), 6.89 (dd, J = 6.6, 2.7 Hz, 1H), 6.46 –
13
.39 (m, 1H), 5.05 (br s, 2H), 2.70 (s, 3H). C NMR (126 MHz, DMSOꢀd ) δ 164.93, 160.84,
6
48.49, 147.72 (d, J = 233.9 Hz), 145.08 (d, J = 1.9 Hz), 137.19, 131.19, 128.44, 128.33, 127.95,
125.50 (d, J = 13.1 Hz), 125.34, 122.99, 115.54 (d, J = 20.6 Hz), 111.52, 111.39 (d, J = 6.6 Hz),
19
+
2
5.05. F NMR (470 MHz, DMSOꢀd ) δ ꢀ138.12. HRMS (ESI ): calcd for C H FN O (M +
6
17 15
3
+
H) , 296.1194; found 296.1191.
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