Improvement of Oral Bioavailability of Pyrazolo-Pyridone Inhibitors of the Interaction of DCN1/2 and UBE2M

Article abstract of DOI:10.1021/acs.jmedchem.1c00035

The cullin-RING ubiquitin ligases (CRLs) are ubiquitin E3 enzymes that play a key role in controlling proteasomal degradation and are activated by neddylation. We previously reported inhibitors that target CRL activation by disrupting the interaction of defective in cullin neddylation 1 (DCN1), a CRL neddylation co-E3, and UBE2M, a neddylation E2. Our first-generation inhibitors possessed poor oral bioavailability and fairly rapid clearance that hindered the study of acute inhibition of DCN-controlled CRL activity in vivo. Herein, we report studies to improve the pharmacokinetic performance of the pyrazolo-pyridone inhibitors. The current best inhibitor, 40, inhibits the interaction of DCN1 and UBE2M, blocks NEDD8 transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above the biochemical IC90 for 24 h in mice.

Full text of DOI:10.1021/acs.jmedchem.1c00035

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Article
Improvement of Oral Bioavailability of Pyrazolo-Pyridone Inhibitors
of the Interaction of DCN1/2 and UBE2M
Ho Shin Kim, Jared T. Hammill, Daniel C. Scott, Yizhe Chen, Amy L. Rice, William Pistel,
Bhuvanesh Singh, Brenda A. Schulman, and R. Kiplin Guy*
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ABSTRACT: The cullin-RING ubiquitin ligases (CRLs) are
ubiquitin E3 enzymes that play a key role in controlling
proteasomal degradation and are activated by neddylation. We
previously reported inhibitors that target CRL activation by
disrupting the interaction of defective in cullin neddylation 1
(DCN1), a CRL neddylation co-E3, and UBE2M, a neddylation
E2. Our first-generation inhibitors possessed poor oral bioavail-
ability and fairly rapid clearance that hindered the study of acute
inhibition of DCN-controlled CRL activity in vivo. Herein, we
report studies to improve the pharmacokinetic performance of the
pyrazolo-pyridone inhibitors. The current best inhibitor, 40,
inhibits the interaction of DCN1 and UBE2M, blocks NEDD8
transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified
squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above
the biochemical IC90 for 24 h in mice.
complete ablation of neddylation appears to be clinically
efficacious,14 global inhibition of neddylation may limit its
therapeutic index. In an effort to maintain efficacy and reduce
toxicity, we sought more subtle ways to regulate the neddylation
pathway.15
INTRODUCTION
■
Neddylation is a post-translational modification that conjugates
the neural precursor cell expressed, developmentally down-
regulated 8 (NEDD8) protein to protein substrates. Neddyla-
tion occurs through a three-step enzymatic cascade. First,
NEDD8 is conjugated to a cysteine of the E1 in an ATP-
dependent manner. Next, a transthioesterification reaction
transfers the NEDD8 to a cysteine on the E2. Finally,
NEDD8’s C-terminus is ligated to the γ-amino group of a lysine
side chain on the target protein forming an isopeptide bond. The
best-known neddylation substrates are the cullins, which upon
neddylation join a multiprotein complex to form cullin-RING
ubiquitin E3 ligases (CRLs). The CRLs are the largest class of
ubiquitin E3 ligases that control ubiquitination of many
proteins. Ubiquitination is a closely related post-translational
modification pathway that regulates many biological processes,
including proteosomal degradation of targets. CRL dysfunction
is implicated in a number of human diseases, including
cancer.1−3
Extensive drug discovery efforts have targeted the CRLs and
the associated proteasomal protein degradation machinery.4−11
The neddylation pathway can be completely inhibited by the E1
inhibitor pevonedistat (MLN4924), which is currently being
investigated in multiple oncology clinical trials.12 NEDD8 is
removed from cullins by the COP9 signalosome (CSN).
Inhibitors of the CSN have also been described and display
promising antitumor activity in animal models.13 While
Defective in cullin neddylation 1 (DCN1) is also known as
DCUN1D1, DCNL1, or squamous cell carcinoma-related
oncogene (SCCRO); we use “DCN1” hereafter. DCN1 acts
as a co-E3 together with RBX1 to stimulate the transfer of
NEDD8 from its E2 (UBE2M) to the cullin proteins, regulating
CRL stability, intracellular localization, and function.16 The
neddylation and ubiquitination pathways are complex dynamic
processes. Thus, the development of efficacious chemical
probes, which permit acute and selective inhibition of DCN-
mediated CRL activity, has the potential to help unravel the
mechanisms regulating key cellular signaling networks and
driving disease progression. In humans, the DCN family
contains five isoforms. The best characterized DCN isoforms
are DCN1 and DCN2, which are highly homologous and may be
redundant in mammals.17−24 They also have the strongest
Received: January 8, 2021
Published: May 4, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00035
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a b
,
Table 1. SAR and SPR of Hinge Pocket
a
IC50 values were generated using our time-resolved fluorescence energy transfer (TR-FRET) binding assay and are represented as the mean of
b
three replicates with errors reported as the standard deviation. Chemical structure and key data for NAcM-OPT.
Figure 1. Design strategy and X-ray co-structure overlay of compound 2:DCN1 (PDB 6P5V) and UBE2M:DCN1 (PDB 3TDU), highlighting the key
residues for interaction and regions targeted for optimization. The key N-terminal residues of UBE2M that control binding to DCN1 are represented in
cyan, compound 2 in orange, and the DCN1 protein as surface in gray with key residues shown as sticks.
associations with disease progression, especially squamous cell
carinomas.17,20,23,25,26
pronounced effects on CRL-based proteasomal degradation
than global neddylation inhibitors targeting the NEDD8 E1, like
MLN4924. This difference arises because the DCN1/2
inhibitors are isoform-selective and only partially inhibit cullin
neddylation. Although the current DCN1 inhibitors are useful
tools for in vitro studies, only the piperidinyl series, represented
by NAcM-OPT (Table 1), are reasonably orally bioavailable in
the mouse.27 However, even high doses of NAcM-OPT (200
We and others have discovered potent and selective inhibitors
of the DCN1/2-UBE2M protein interaction.27−33 These
inhibitors all bind to and thermally stabilize DCN1 in cells
and selectively reduce the steady-state levels of cullin
neddylation in a variety of cell lines including HCC95,
CAL33, KYSE70, H2170, SK-MES-1, and MGC-803
cells.27−33 As a class, the DCN1/2 inhibitors have less
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Scheme 1. Synthesis of Pyrazole Methyl-Modified DCN1 Inhibitors
mg/kg, BID) do not continuously maintain plasma concen-
trations at or above its cellular IC90.27
compound 2, is potent and selective but only moderately soluble
and stable in microsomal models. Compound 2 does not address
the key liability of NAcM-OPTthe poor pharmacokinetics
hindering the use for modeling in vivo. To address this issue, we
embarked on a second medicinal chemistry campaign to develop
chemical probes of pyrazolo-pyridone class useful for in vivo
studies.
We recently disclosed the pyrazolo-pyridones, a class of
DCN1 inhibitors designed to address several limitations of
NAcM-OPT including: (1) failure to access the N-acetyl
subpocket in the targeted binding pocket that controls the
binding of the native substrate; (2) a minimal three-dimensional
character, limiting efficient access of the available binding
subpockets; and (3) the moderate murine half-life and Cmax that
drive the poor pharmacokinetic profile.33 Our initial work
focused on addressing the first two points, identifying the
minimum pharmacophore for this class, and defining the
structural drivers of potency for binding to DCN1. Briefly,
these studies yielded compound 2, which was 25-fold more
potent than the original HTS hit compound. Compound 2
design was based on insights derived from overlaying the X-ray
co-structures of the HTS hit:DCN1 (PDB 6P5W),
UBE2MNAc:DCN1 (PDB 3TDU), and NAcM-OPT:DCN1
(PDB 5V86).
The previous SAR studies revealed four critical components:
(1) a strong preference for the cis stereochemistry of the
pyrazolo-pyridones; (2) a preference for small alkyl substitution
of the pyridone nitrogen; (3) wide flexibility with respect to the
identity of substituents fitting either Ile or Leu pocket, but the
potential for metabolic degradation; and (4) very restrictive
requirements for the substituent targeting the hinge pocket
(Figure 1). The best lead compound arising from those studies,
Herein, we summarize the key learnings from this campaign in
which we prepared and analyzed over 150 new analogues
designed based on the hypotheses generated by combining
empirically derived SAR/SPR from earlier studies with
examination of X-ray co-structures. New analogues were tested
for their ability to inhibit the target protein interaction using our
previously reported TR-FRET assay based on the TR-FRET
signal between a biotinylated DCN1 protein, recognized by
Terbium-linked streptavidin, and the helical stapled peptide
derived from N-terminally acetylated UBE2M harboring a C-
terminal AlexaFluor 488.29 Active and potent compounds were
evaluated for biochemical inhibition of neddylation in a
reconstituted pathway cascade, cellular target engagement, and
inhibition of anchorage-independent growth in a DCN1
amplified cell line (HCC95). In parallel, active compounds
were tested to evaluate the solubility and stability to incubation
with liver microsomes. Select molecules were subjected to
pharmacokinetic studies in mice. Ultimately, these studies
identified compound 40, for which a single 50 mg/kg oral dose
affords continuous murine plasma exposure above its bio-
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Scheme 2. Parallel Synthesis of Analogues Targeting the Hinge Pocket
chemical IC90 for at least 24 h. Compound 40 was also slightly
(2-fold) more potent than compound 2 at inhibiting the DCN1-
UBE2M interaction, thermally stabilizes DCN1 in cells, and
inhibits anchorage-independent growth of transformed cells.
accomplished via either Appel reaction and substitution or
Gabriel synthesis. This route, while efficient, was not amenable
to rapid diversification of the hinge pocket. For this purpose, we
modified the synthetic route to include amide hydrolysis,
followed by either acylation with an acid chloride/anhydride or
EDCI-mediated amide coupling (Scheme 2). Unless otherwise
specified, all compounds were purified using flash column
chromatography and their purity (>95%) and identity were
RESULTS
■
Design and Synthesis. The pyrazolo-pyridones were
discovered during our initial high-throughput screening
campaign (HTS)29 and optimized for potency to give
compound 2.33 Relative to the first class of inhibitors reported
by our group, as exemplified by NAcM-OPT33 the pyrazolo-
pyridones possess a high degree of three-dimensional structure
that affords opportunities for increased binding potency, target
selectivity, and improved solubility. The structure-guided
optimization that afforded 2 defined four key subpockets within
the binding site: Ile, Leu, N-acetyl, and hinge pockets (Figure 1).
The most important learning from these studies was that ethyl
substitution of the pyridone nitrogen could induce tighter
binding by enhanced hydrophobic packing compared to the
indirect water-mediated binding mode seen with unsubstituted
pyridones. Although compound 2 is potent and selective, it is
also poorly water-soluble and readily metabolized leading to
poor bioavailability. We embarked on the second medicinal
chemistry campaign to improve the bioavailability of compound
2.
1
established by liquid chromatography, H NMR, and HRMS
analyses. Key analogues were further characterized by 13C
NMR.
Reagents and conditions: (a) (i) CH3CN, n-BuLi, ethyl
glycolate, tetrahydrofuran (THF), −78 °C to room temperature
(rt), 2h, (ii) phenyl hydrazine, chlorobenzene, reflux, 16 h; (b)
chlorobenzene, 170 °C, in pressure vessel, 16 h; (c) EtBr,
Cs2CO3, dimethylformamide (DMF), rt, 16 h; (d) HCl,
CH3CN, rt, 16 h; (e) CBr4, PPh3, dichloromethane (DCM), 0
°C to rt, 16 h; (f) R1R2N, N,N-diisopropylethylamine (DIPEA),
DCM, 0 °C to rt, 16 h; (g) (i) phthalimide, K2CO3, DMF, rt, 16
h, (ii) hydrazine monohydrate, DCM, 0 °C to rt, 16 h; (h) Ac2O
or MsCl or R-COCl, DIPEA, DCM, 0 °C to rt, 1 h.
Reagents and conditions: (a) c-HCl, 1,4-dioxane, 85−90 °C,
16 h; (b) R-COOH, EDCI·HCl, DCM, rt, 16 h; (c) R-COCl,
DIPEA, DCM, rt, 16 h.
Structure−Activity and Structure−Property Relation-
ships. In the course of this study, we designed, prepared, and
tested over 150 analogues. To provide a clearer narrative, we
show in the main text only critical compounds illustrating the
key structural drivers for potency (structure−activity relation-
ships, SAR); permeability, solubility, and log D (structure−
property relationships, SPR); and metabolic and toxic liabilities.
Complete SAR and SPR tables, containing all compounds made
and tested, can be found in the Supporting Information.
We began the campaign by validating the hypothesis that
previously observed structural drivers of potency in the absence
of pyridone substitution are maintained with a substituted
pyridone.33 This was done to ensure that there were no
nonadditive interactions between the N-ethyl and other
substituents that dramatically shifted SAR.
First, we sought to confirm the importance of the relative
stereochemistry of the pyrazolo-pyridone ring. Surprisingly,
some trans analogues, which in the absence of pyridone
substitution were universally inactive (>15 μM), exhibited
moderate activity. In some cases, they were even as potent as
their cis counterparts. However, not all trans analogues exhibited
activity (Table S10). Therefore, we maintained focus on the cis
diastereomers for all future work and to enable better
comparison to legacy data.
Our overall goal was to achieve a 10-fold improvement in
plasma exposure (AUC) while maintaining or improving
potency. Thus, we set out to: (1) reduce in vivo clearance by
suppressing oxidative metabolism; (2) improve aqueous
solubility and maximal plasma exposure by reducing hydro-
phobicity and crystallinity; (3) refine the pharmacophore by re-
visiting favored substituents for the Ile, Leu, and Hinge pockets
within the context of the cis-ethyl pyridone; and (4) expand the
range of N-acetyl pocket substituents with hydrophilic
substitutions that may form beneficial electrostatic or hydro-
gen-bonding interactions within the N-acetyl pocket.
New compounds were synthesized using a modification of our
previously reported three-step procedure (Scheme 1)33
consisting of: preparation of an oxazolone intermediate,34
pyrazolo-pyridone ring formation,35 and substitution using
alkylation or acylation. Formation of the core pyrazolo-pyridone
ring afforded a separable mixture of cis- and trans- diastereomers,
which were assigned based on the 3JH−H vicinal proton−proton
coupling for the C4 or C5 protons of pyrazolo-pyridone ring (cis
= 7−8 Hz, trans = 9−11 Hz). Focused derivatization of the
pyrazole methyl began with displacement of the ethoxy group of
silyl-protected ethyl glycolate with acetonitrile. Subsequent
reaction with various hydrazines afforded the key 5-amino-3-
silyl-protected hydroxyl pyrazole intermediates. Cyclization of
the amino pyrazole with an oxazolone, ethylation of the
pyridone, and silyl deprotection produced free hydroxylated
compounds, generally in moderate yields. The introduction of
structural diversity on the solvent-exposed methyl pyrazole was
Next, we sought to confirm the SAR about the hinge region.
Similar to the unsubstituted pyridone SAR,33 substituents
targeting the hinge pocket maintained structural requirements,
requiring a small hydrophobic substitution such as a meta-
methyl group for maximal potency. Removing all substitution
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from the aryl ring (1) decreased the potency 6-fold.
Replacement of the methyl group with roughly isosteric
electron-withdrawing groups was well tolerated (CF3 (4), Br
(48), I (49), Table S1). Significantly smaller or larger
substituents decreased potency. Smaller heterocyclic replace-
ments of the phenyl ring were tolerated in some, but not all cases
(7, 8, Table S2), with no clear trends beyond the steric fit. Polar
substituents (10, 11, 71, 72; Tables S1 and S2) were not favored.
Adding fluorine atoms was generally tolerated, but decreased
potency relative to nonfluorinated analogues (Tables 1, 2 vs 4
min/kg, 5.2 L/h/kg). Maximal plasma exposure (Cmax = 0.36
μM) at 50 mg/kg oral dose was also limited. Overall, there was a
good correlation between predicted clearance from microsomal
models and observed clearance. To guide the design of
metabolically stable analogues, we conducted metabolite
identification (Met-ID) studies for compound 2 after micro-
somal incubation, revealing that oxidation of the aryl amide
substituent targeting the hinge pocket was the major route of
metabolism. Electronically and physically deactivating the site of
metabolism by introducing halogenated substituents signifi-
cantly decreased the rate of metabolic oxidation (3, 4, Table 1
and Figure 2), while electron-donating substituents made the
compounds more susceptible to oxidative metabolism (OCH3,
50, Table S1). Since the meta-CF3 phenyl ring afforded the best
combination of potency, solubility, and oxidative metabolic
stability (4, Table 1 and Figure 2), we anchored further studies
upon that substituent and began to explore the N-acetyl pocket.
Our initial SAR study33 included a systematic exploration of
substituents targeting the N-acetyl pocket. That study revealed
that ethyl substitution of the pyridone most effectively occupied
the N-acetyl binding pocket on DCN1 and improved potency 5-
to 10-fold, relative to nonethylated analogues. Previous
biochemical and crystallography studies have shown that
occupancy of this pocket on DCN1 by the native acetylated
N-terminus of UBE2M is critical for the protein interaction and
stabilized by both hydrophobic fulfillment of the pocket and
hydrogen bonding between the N-terminal amide of UBE2M
and two key residues on DCN1: the donor hydroxyl of Tyr181
and acceptor amide backbone of Pro97 (PDB 3TDU).25
Therefore, we revisited the N-acetyl pocket with a focus on
the introduction of substituents including hydrogen-bond
donors and acceptors. While we surveyed almost 20 substituents
(Table S3), only the ethyl hydroxy (19) was comparably potent
to the ethyl analogue (4). Other groups including the lipophilic
F (83), CN (21), and hydrophilic NH2 (20) were not tolerated
(Tables 2 and S3). Although the hydroxyl (19) is polar and
retains metabolic stability, it did not improve aqueous solubility.
The combination of data suggests a strict steric requirement,
which is further supported by the analysis of the X-ray co-crystal
structure (Figure 1). A brief survey of substituents targeting the
Ile pocket revealed that replacement of the phenyl ring with a
small aliphatic group (22, 24) was well tolerated in terms of
potency but led to a reduction in metabolic stability (Table S4).
Fluorinated substituents, meant to suppress n-dealkylation, were
not tolerated (23, 26, Table 2).
Next, we focused on further exploration of substituents
targeting the Leu subpocket. We explored replacing one of the
three aryl rings that cause the compounds high log D36 with
heterocycles designed to improve water solubility. Some
heterocyclic analogues retained potency (27−29, Table 3).
Among these, placing a sulfur atom at the 3-position proved
critical for potency (27−29). However, placement of heter-
oatoms at the 2-position led to a 5-fold drop in potency (Table
S5). Within the subset of active heterocycles, incorporation of a
methyl group at either the 2- or 3-position was well tolerated
(28, Table S5). Unfortunately, these heterocycles did not
improve either solubility or microsomal stability.
We turned to a wider exploration of other substituents that
might improve either solubility or metabolic stability. Fluorina-
tion at the para-position of the phenyl ring (4) proved optimal,
and larger substituents reduced potency (vs 31). Consistent
with previous studies,33 the presence of a para-F preserved the
activity of the pyridine rings (33). However, the para-
a
Table 2. SAR of N-Acetyl Pocket and IIe Pocket
a
IC50 values were generated using our TR-FRET binding assay and
are represented as the mean of three replicates with errors reported as
the standard deviation.
and S1). Any significant increase in steric bulk was poorly
tolerated. Bicyclic replacement (12) or mimicking the optimized
hinge pocket of NAcM-OPT (9) significantly reduced potency.
Aliphatic substitution (13), elongation of the linker (14), or
isosteric replacement of the linker (15, Table S2) were not
tolerated. Throughout this study, analogues containing ethyl
substitution of the pyridone were universally more potent than
their unsubstituted counterparts, consistent with the key SAR
findings of our previous publication.33 Clearly, there remains a
strict steric requirement for this substituent that does not permit
size modifications without loss of potency. Taken together, these
two preliminary studies confirmed overall maintenance of the
previously defined SAR.
The major liabilities identified during the first round of
optimization were poor aqueous solubility and moderate
oxidative metabolic stability.33 To assess if the microsomal
models were accurately predicting in vivo clearance, we tested
compound 2 in single-dose intravenous (iv) and oral
pharmacokinetic (PK) studies (Figures 2 and S6). We did not
observe any adverse reactions or compound-related side effects
within 48 h after dosing. Compound 2 was poorly oral
bioavailable (15%) and rapidly cleared both in vitro and in
vivo (intrinsic MLM CLint = 44.9 mL/min/kg, predicted in vivo
hepatic CL = 32.7 mL/min/kg, murine plasma CLIV = 86.5 mL/
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a
improve aqueous solubility. Based on X-ray co-crystallography
analysis (Figure 1, PDB 6P5V, 3TDU), we hypothesized that the
pyrazole methyl was pointing to solvent and would be tolerant of
such modifications. Indeed, the introduction of polar sub-
stituents at this position increased solubility and modestly
improved potency (2- to 4-fold). Even larger substituents with
longer chains (46, Table 4) were tolerated here. However, if
substantial steric demand is introduced too close to the interior
of the binding pocket, it can substantially reduce potency (38 vs
46, Table 4). Oxidation of the methyl to the carboxylic acid
reduces potency 3-fold (39, Table 4). Compared to the hydroxyl
Table 3. SAR and SPR of Leu Pocket
a b
,
Table 4. SAR and SPR of New Pocket
a
IC50 values were generated using our TR-FRET binding assay and
are represented as the mean of three replicates with errors reported as
the standard deviation.
fluoropyridine analogue was not significantly more soluble than
the thiophene. Other substituents at the meta-position reduced
potency except the nitro group (35). Halogenation at ortho-
position was well tolerated (Tables 3, S5 and S6). Finally, we
explored the effect of increasing total rotatable bonds while
reaching deeper into the Leu subpocket using the benzyl
replacement for phenyl. The benzyl reduced potency 2-fold
compared to its para-fluorophenyl analogues (4, 32, Table 3).
While metabolic stability was greatly improved by fixing the
meta-CF3 phenyl as the substituent fulfilling the hinge pocket,
this modification increased compound lipophilicity (C log P 4 =
5.26) and decreased solubility. Thus, we sought to introduce a
polar ionizable group that could decrease the lipophilicity and
a
IC50 values were generated using our TR-FRET binding assay and
are represented as the mean of three replicates with errors reported as
b
the standard deviation. Compounds 40a (faster eluting) and 40b
(slower eluting) are the pure enantiomers of 40, which were separated
by chiral SFC.
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Figure 2. Pharmacokinetic profiling of compounds 2, 4, and 40. (A) Time course of the drug plasma concentration over 24 h resulting from single-dose
intravenous administration of compounds at 15 mg/kg. (B) Time course of the drug plasma concentration over 24 h resulting from single oral dose of
compounds at 50 mg/kg. aCalculated up to 96 h time point (not fully cleared at that time). Any values of concentration below LLOQ (dotted line)
were reported as half LLOQ.
analogue (37), the amino (40) or substituted amino compounds
(41−46, 103−123, Tables S7−S9) showed equivalent or
slightly better potency (ca. 2-fold) regardless of basicity. Of
the substitutions studied, primary amine 40 had the best balance
of properties including good potency (100 nM), good aqueous
solubility (71 μM), and excellent oxidative metabolic stability
(MLM Clint <10 mL/(min kg)). The enantiomers of
compound 40 were separated to ca. 99% enantiomeric excess
(ee) by chiral SFC using an IG column. The faster eluting
enantiomer (40a) was more potent (IC50 = 0.06 μM) than the
slower eluting enantiomer (40b; IC50 = 10 μM). All further
modification of the primary amine compromised microsomal
stability and/or solubility (Tables S7−S9). To further improve
water solubility, we prepared both the phosphate and hydro-
chloride salts. Both salts significantly improved solubility with
the phosphate salt affording a 10-fold boost in solubility (668
μM).
To test our hypothesis that murine exposure could be
improved by mitigating oxidative metabolism and increasing
solubility, we selected three compounds (2, 4, 40) for murine
PK modeling (Figure 2). Each compound was dosed
independently by intravenous and oral routes after being
formulated in EtOH/PG/PEG/PBS (10/10/40/39; v/v/v/v;
pH = 7.4) and 1% (w/v) 2-hydroxy-β-cyclodextrin. As briefly
discussed above, a single oral 50 mg/kg dose of 2 afforded low
methylamine substitution to the pyrazole ring in this back-
ground (40) greatly improved total plasma exposure (50 mg/kg
PO, AUC = 72.9 h·μM Cmax = 6.0 μM), dramatically enhanced
oral bioavailability (92%), and maintained minimal clearance
(plasma ClIV = 1.2 L/(kg h)) (Figure 2). Compound 40 was also
well tolerated at these doses with no significant changes
observed in clinical chemistry, hematology, or signs monitored
during a functional observational battery (Tables S16 and S17,
Figures S9 and S10). These experiments established an in vitro−
in vivo correlation (IVIVC) for the series between microsomal
stability models, solubility, and in vivo bioavailability. This
allows use of in vitro models to rapidly triage and prioritize new
molecules for in vivo testing in the future (Figures 2 and S6−
S8).
Biochemical and Cellular Profiling of Compound 40.
Having identified compound 40 with significantly improved
murine pharmacokinetic performance and slightly improved
affinity, we carried out functional assessments to ensure it
remained on target and active in cells. Compound 40 was
equally potent in our assay that monitors the transfer of a FAM-
labeled NEDD8 to cullin substrates in a fully reconstituted
NEDD8 cascade (IC50 = 0.11 μM, Figure S1),16,29,37
demonstrating that the increased potency for blocking DCN1-
UBE2M binding effectively translates to inhibition of
neddylation. Importantly, compound 40, and several other
pyrazolo-pyridone analogues, showed a more rapid on-rate and
slower dissociation rate than our original leads, demonstrating
increased drug−target residence time compared to NAcM-OPT
in surface plasmon resonance (SPR) assays (5- to 37-fold, Table
S2 and S3). Slow dissociation of 40 suggests that the duration of
occupancy of the intracellular DCN1−inhibitor complex is
extended, which may boost efficacy and lengthen the duration of
the effect.38
overall plasma exposure (50 mg/kg PO, AUC = 2.9 h·μM, Cmax
=
0.36 μM) and poor oral bioavailability (15%). We hypothesized
that the low exposure and bioavailability were driven by its poor
water solubility (16 μM) and fast oxidative metabolism (plasma
ClIV = 5.2 L/(kg h)).
Replacement of the toluyl ring targeting the hinge pocket (2)
with the m-CF3 phenyl (4) significantly improved in vivo murine
clearance (plasma ClIV = 0.64 L/(kg h); Figure 2). However, the
oral plasma exposure of compound 4 (50 mg/kg PO, AUC =
23.8 h·μM, Cmax = 1.15 μM) and oral bioavailability (36%)
remained relatively low. This supported the hypothesis that
clearance is predominantly driven by oxidative metabolism and
strongly suggested that poor absorption driven by its low
solubility (11 μM, Table 1) was also playing a role. Adding the
Having established the expected biochemical performance,
we examined if the improvement in PK came at the expense of
cellular potency. Compound 40 clearly maintains target
engagement in a squamous cell carcinoma (SCC) cell line that
contains DCN1 amplification (HCC95) as demonstrated with a
cellular thermal shift assay (CETSA).39 The DCN1 protein is
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largely degraded at 52 °C in HCC95 cells treated with dimethyl
sulfoxide (DMSO). However, in the presence of 10 μM of
compound 40, or the known DCN1/2 inhibitor NAcM-OPT,15
the thermal stability of the DCN1 protein is clearly enhanced.
Gratifyingly, it also appears that the improvement in
biochemical potency and drug−target residence time translates
to increased cellular target engagement as demonstrated by
enhanced DCN1 stabilization in the CETSA assay (Figure 3)
CETSA assays (Figure S4). At this concentration, none of the
compounds are growth inhibitory to HCC95 (EC50 > 24 μM,
data not shown). Additionally, none of the tested compounds
inhibit the proliferation of normal fibroblasts (BJ; ATCC CRL-
2522) up to the maximum tested concentration of 24 μM (data
not shown).
DISCUSSION AND CONCLUSIONS
■
The overall goal of this study was to determine if this lead series
could deliver potent inhibitors of the DCN1-UBE2M
interaction that were also highly orally bioavailable and
possessed reasonable to long in vivo half-life such that they
were suitable for interrogating acute pharmacologic inhibition of
DCN1/2-mediated cullin neddylation in murine models.
Herein, we coupled a rational, structure-based design with
empirical exploration of substituents to uncover the key
structural elements critical for potency, solubility, and metabolic
stability. The efficient synthetic routes outlined in Scheme 1
enabled rapid access to over 150 analogues to define the SAR/
SPR and permitted multi-gram-scale preparation of several key
analogues. Ultimately, these studies identified compound 40,
which inhibits DCN1-UBE2M binding, blocks DCN1-mediated
cullin neddylation, engages cellular DCN1 (Figure 3), and
restores contact inhibition to HCC95 cells (Figure 4). Most
importantly, 40 has a 25-fold improved plasma exposure, relative
to compound 2, and excellent oral bioavailability such that a
single 50 mg/kg oral dose of 40 affords continuous murine
plasma concentrations above its biochemical IC90 (Figure 2) for
24 h.
Figure 3. Enhancement of DCN1 thermal stability by compounds 40
and NAcM-OPT (positive control) but not by DMSO (negative
control). HCC95 cells were treated with either DMSO or 10 μM of the
indicated compound for 1 h, heated at the indicated temperature for 3
min, lysed, and blotted with the indicated antibodies.
relative to NAcM-OPT. Dose−response experiments (Figure
S4) suggest an approximate cellular EC50 between 0.3 and 1 μM,
consistent with the biochemical IC90 in the binding and
functional assays.
Human DCN1 was originally discovered due to its over-
expression in tumor cells, with amplification conferring
anchorage-independent growth.40 We have previously demon-
strated that inhibitors of the DCN1 and UBE2M interaction
restore anchorage dependency as scored in a soft-agar assay.
Compound 40 inhibited colony formation completely at a
concentration of 10 μM in this assay (Figure 4), showing that the
This study had three major findings (Figure 5). First, the
incorporation of a small hydrophobic ethyl substitution on the
pyridone proved superior to hydrophilic substitutions and
restored the activity of some of the trans diastereomers that had
been previously universally inactive. Second, the methyl
pyrazole affords access to an electronically and sterically tolerant
primarily solvent-exposed subpocket. Substitution of the methyl
pyrazole with ionizable polar substituents dramatically improved
aqueous solubility without negatively affecting binding to the
target or activity. Third, we established an in vitro−in vivo
correlative model between microsomal stability and solubility
and in vivo bioavailability that allowed us to confidently select
optimized compounds for in vivo modeling. Metabolite
identification studies after microsomal incubation focused our
attention on the most rapidly metabolized moieties of the
inhibitor series: the aryl ring targeting the hinge pocket.
Electronic and steric deactivation of the aryl ring via the
incorporation of the CF3 substituent effectively suppressed
oxidative metabolism in microsomal and murine models.
This new understanding of the determinants of bioavailability
and potency led to the discovery of the optimized compound 40
that possesses greatly improved in vivo murine PK (oral
bioavailability, Cmax, IV Clp, and AUC) and strong effects in
cellular assays. Improvement in inhibitory potency for the
DCN1-UBE2M protein−protein interaction (TR-FRET) and
drug−target residence time (SPR) translated well to inhibitory
potency in preventing biochemical neddylation in the presence
of the entire CRL complex (pulse-chase). In cellular models,
compound 40 shows stronger thermal stabilization of the DCN1
protein relative to previous compounds, demonstrating that 40
effectively engages DCN1 in cells and that the alterations to its
physiochemical properties did not significantly compromise
permeability. Finally, compound 40 strongly inhibits anchorage-
Figure 4. Soft-agar colony formation assay of DMSO, NAcM-OPT, 2,
and 40. HCC95 cells were treated with either DMSO or 10 μM of the
indicated compound. Colonies were counted at 10 days post-treatment.
The experiment was repeated three times. Each data point is the average
of colonies in three wells per one biological replicate.
expected phenotypic response is exhibited at concentrations
where there is strong target engagement. Compound 40 is
equally efficacious to NAcM-OPT at this concentration and
significantly more efficacious than the early lead in the pyrazolo-
pyridone series, compound 2. Dose−response experiments
(Figure S5) further support an approximate cellular EC50
between 0.3 and 1 μM, consistent with the dose−response
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Figure 5. SAR, SPR summary, and X-ray crystal structure compound 40 bound to DCN1 (PDB ID: 7KWA).
1
SAR analysis were further characterized using H NMR and HRMS/
LRMS.
independent colony formation of HCC95 cells, showing the
primary expected phenotypic cellular response.
General Procedure. A. Pyrazole Cyclization. A solution of
acetonitrile (1.5 mmol) in dry THF (2.0 mL) was cooled to −78 °C,
and 2.5 M n-BuLi in hexanes (1.5 mmol) was added dropwise. The
reaction mixture was stirred for 15 min at −78 °C, and ethyl 2-((tert-
butyldimethylsilyl)oxy)acetate (1.0 mmol) was added. The reaction
mixture was allowed to warm to ambient temperature and was stirred
overnight. The reaction mixture was diluted with ice water. The residual
aqueous mixture was acidified to pH = 5 and extracted with EtOAc. The
combined organics were washed with brine, dried over MgSO4, filtered,
and concentrated.
To a solution of the intermediate in chlorobenzene (1.0 mL),
hydrazine (1.0 mmol) was added and the reaction mixture was refluxed
overnight. Upon cooling, the mixture was treated with saturated
NaHCO3 until basic and the mixture was extracted with CH2Cl2. The
organic layer was dried over MgSO4, filtered, and concentrated. The
mixture was purified by flash chromatography.
B. Oxazolone Key Intermediate with Aliphatic Chain. B.1. Glycine
Coupling. Carbonyl chloride (1.0 mmol) was added to a stirred
solution of glycine (1.0 mmol) in 1 N aqueous sodium hydroxide (3.0
mL) dropwise. The reaction mixture was stirred at room temperature
overnight. Then, the pH of the mixture was adjusted to 1−2 with 1 N
aqueous HCl. The resulting solution was extracted with CH2Cl2, dried
over MgSO4, filtered, concentrated under reduced pressure, and
purified by flash chromatography.
B.2. EDC Cyclization. Under a nitrogen atmosphere, the amine (1.0
mmol), EDCI·HCl (1.3 mmol), and DIPEA (1.3 mmol) were added to
a solution of carboxylic acid (1.0 mmol) in CH2Cl2 (3 mL). The
reaction mixture was stirred at room temperature overnight. The
resulting solution was extracted with CH2Cl2, dried over MgSO4,
filtered, concentrated under reduced pressure, and purified by flash
chromatography.
In conclusion, compound 40 is sufficiently potent and orally
bioavailable to permit acute pharmacologic regulation of this
highly complex and dynamic pathway in murine models. Ideally,
this tool compound will enable the study of previously
unappreciated biological roles of DCN1-mediated neddylation
and provide further insights into DCN1/2’s role in tumor
progression. We are currently using compound 40 and others to
dissect the effects of acute pharmacologic inhibition of the
DCN1-UBE2M interaction on the NEDD8/CUL pathway in
murine models and will report those results in due course.
EXPERIMENTAL SECTION
■
TR-FRET Assay. The TR-FRET assay was carried out in black 384-
well microtiter plates (Corning 3573) at a final volume of 20 μL per
well. The assay cocktail was prepared as a mixture of 50 nM biotin-
DCN1, 20 nM Ac-UBE2M12-AlexaFluor 488, and 2.5 nM Tb
streptavidin (Thermo Fisher) in assay buffer (25 mM HEPES, 100
mM NaCl, 0.1% Triton X-100, 0.5 mM DTT, pH 7.5). The assay
cocktail was then incubated in bulk for 1 h at room temperature and
then distributed (20 μL per well) using a WellMate instrument
(Matrix). Compounds to be screened were added to assay plates from
DMSO stock solutions by pin transfer using a pin tool (AFIX384FP1,
V&P Scientific) adapted for manual transfer (BGPK, VP 381D-N, V&P
Scientific) and equipped with 50SS pins (V&P Scientific). The assay
plates were incubated for 1 h at room temperature prior to measuring
the TR-FRET signal with a Clariostar plate reader (BMG Labtech)
equipped with excitation maxima at 337 nm and emission maxima at
490 and 520 nm. We set the integration start to 100 μs and the
integration time to 200 μs. The number of flashes was set to 100. The
relative fluorescence (Ex/Em = 520:490) was used for TR-FRET signal
calculations. Assay end points were normalized from 0% (DMSO only)
to 100% inhibition (unlabeled competitor peptide) for hit selection and
curve fitting. All compounds were tested in triplicate or more.
B.3. Al2O3 Condensation. Under a nitrogen atmosphere, the
aldehyde (0.5 mmol) was added dropwise to a suspension of oxazolone
(0.1 mmol), activated molecular sieves 4 Å (1.0 g), and activated
aluminum oxide (1.0 mmol) in anhydrous CH2Cl2 (3 mL). The
reaction mixture was stirred at room temperature for 6 h. The resulting
solid was filtered through a pad of Celite to remove molecular sieves and
Al2O3. The filtrate was dried under reduced pressure and then purified
by flash chromatography.
Chemistry Experimental. General. All NMR data were collected
at room temperature in CDCl3 or (CD3)2SO on a 400 or 500 MHz
Bruker or Agilent instrument. Solvents and reagents were used directly
as obtained from commercial sources unless otherwise specified.
Chemical shifts (δ) are reported in parts per million (ppm) with
C. Dihydropyridinone Cyclization. The reaction was performed
under either condition (a) or (b).
1
internal CHCl3 (δ 7.26 ppm for H and 77.0 ppm for 13C), internal
(a) Under a nitrogen atmosphere, oxazolone (0.5 mmol) and tin
(II) chloride (0.05 mmol) were added to a solution of amine
(0.5 mmol) in chlorobenzene (1.0 mL). The reaction mixture
was refluxed overnight. After cooling to room temperature, the
mixture was extracted with EtOAc, washed with brine, dried
over MgSO4, filtered, concentrated under reduced pressure, and
purified by flash chromatography.
DMSO (δ 2.50 ppm for 1H and 39.5 ppm for 13C), or internal TMS (δ
0.0 ppm for 1H and 0.0 ppm for 13C) as the reference. 1H NMR data are
reported as follows: chemical shift, multiplicity (s = singlet, bs = broad
singlet, d = doublet, t = triplet, q = quartet, p = pentet, sext = sextet, sep
= septet, m = multiplet, dd = doublet of doublets, ddd = doublet of
doublet of doublets, dt = doublet of triplets, td = triplet of doublets, tt =
triplet of triplets, qd = quartet of doublets), coupling constant(s) (J) in
Hertz (Hz), and integration. Flash column chromatography was
performed using a Biotage Isolera One and Biotage KP-SIL SNAP
cartridges. Purity was assessed by LC/MS/UV using a Waters Acquity
UPLC-MS and by NMR spectroscopy. All compounds were confirmed
to ≥95% purity prior to testing. Compounds that proved critical to our
(b) Under a nitrogen atmosphere, the oxazolone (0.5 mmol) was
added to a solution of the amine (0.5 mmol) in chlorobenzene
or a,a,a-trifluorotoluene (0.5 mL). The mixture was reacted at
150−180 °C in a sealed tube overnight. After cooling, the
mixture was concentrated and the residue was precipitated in
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hexane and filtered. Then, the crude product was purified by
flash chromatography.
overnight. The mixture was extracted with EtOAc, washed with
brine, dried with MgSO4, filtered, and dried. Then, the crude
product was purified by flash chromatography.
D. Dihydropyridinone-amide Substitution. D.1. Dihydropyridi-
none-amide Alkylation. Under a nitrogen atmosphere, cesium
carbonate (1.1 mmol) and the alkyl halide (1.1 mmol) were added to
a solution of dihydropyridinone (0.1 mmol) in DMF (1 mL). The
reaction mixture was stirred overnight at room temperature or heated at
100 °C for 3 h. Then, the mixture was extracted with EtOAc, washed
with brine, dried over MgSO4, filtered, concentrated under reduced
pressure, and purified by flash chromatography.
(b) To a solution of amine intermediate (1.0 mmol) in CH2Cl2 (1.0
mL), carbonyl chloride (1.0 mmol) and DIPEA (1.0 mmol)
were added, and the mixture was stirred at room temperature for
3 h. The mixture was extracted with CH2Cl2, washed with brine,
dried with MgSO4, filtered, and dried. Then, the crude product
was purified by flash chromatography.
Spectral Characterization of Key Compounds. N-(rel-(4S,5S)-
7-Ethyl-4-(4-fluorophenyl)-3-methyl-6-oxo-1-phenyl-4,5,6,7-tetra-
hydro-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-methylbenzamide (Com-
pound 2, Compound Characterization Agreed with Previous
Reports).33 Compound 2 was synthesized by general procedures B−
D starting from 4-fluorobenzaldehyde, (3-methylbenzoyl)glycine, and
3-methyl-1-phenyl-1H-pyrazol-5-amine.
D.2. Dihydropyridinone-amide Acylation. Under a nitrogen
atmosphere, pyridine (0.075 mmol) and acyl chloride (0.075 mmol)
were added to a solution of dihydropyiridinone (0.05 mmol) in CH2Cl2
(1.0 mL). The reaction mixture was stirred at room temperature
overnight. Then, the mixture was extracted with EtOAc, washed with
brine, dried over MgSO4, filtered, concentrated under reduced pressure,
and purified by flash chromatography.
E. Silyl Deprotection. To a solution of silyl-protected compound (1
mmol) in ACN (2 mL) under nitrogen atmosphere, 2 N HCl (3 mmol)
was added and stirred at room temperature overnight. Then, the
mixture was concentrated by reduced pressure, neutralized with sat.
NaHCO3 solution, extracted with CH2Cl2, washed with brine, dried
with MgSO4, filtered, and dried. The mixture was purified by flash
chromatography.
F. Bromination (Appel Reaction). Carbon tetrabromide (1.7 mmol)
was added portionwise to a solution of hydroxy compound (1.0 mmol)
and triphenylphosphine (1.7 mmol) in CH2Cl2 (1 mL) that had been
cooled to 0 °C and placed under nitrogen. The reaction was allowed to
warm to rt and stirred for 2 h. The reaction mixture was filtered and
washed with DCM. The filtrate was concentrated under reduced
pressure. The crude product was purified by flash chromatography.
G. Gabriel Synthesis. To a solution of phthalimide (1.2 mmol) and
potassium carbonate (1.2 mmol) in DMF (0.5 mL), bromo compound
(1.0 mmol) in DMF (0.5 mL) was added at room temperature under
nitrogen. The reaction was stirred overnight. The reaction mixture was
extracted with EtOAc, washed by brine and water, dried over MgSO4,
and then filtered. The filtrate was concentrated under reduced pressure.
The crude product was purified by flash chromatography.
To a solution of phthalimide-protected compound (1.0 mmol) in
MeOH (1 mL) under nitrogen atmosphere, hydrazine monohydrate (3
mmol) was added and stirred at room temperature overnight. After
concentrating and cooling, the mixture was filtered and washed by
CH2Cl2 several times, and then the filtrate was purified by flash
chromatography.
H. Amino Substitution. To a solution of bromo compound (1.0
mmol) in DCM (1.0 mL) under nitrogen atmosphere, amino
compound (R1R2NH) (1.0 mmol) and DIPEA (1.1 mmol) were
added and the reaction mixture was stirred at room temperature
overnight. The mixture was extracted with CH2Cl2, washed with brine,
dried with MgSO4, filtered, and dried. Then, the crude product was
purified by flash chromatography.
I. Acylation at Amino Group. To a solution of amino compound
(1.0 mmol) in CH2Cl2 (1.0 mL) under nitrogen atmosphere at 0 °C,
Ac2O, MsCl, or R−COCl (1.0 mmol) and DIPEA (1.1 mmol) were
added and the mixture was stirred at room temperature 1 h. The
mixture was extracted with CH2Cl2, washed with brine, dried with
MgSO4, filtered, and dried. Then, the crude product was purified by
flash chromatography.
1H NMR (400 MHz, chloroform-d) δ 7.57 (s, 1H), 7.55−7.42 (m,
6H), 7.35−7.28 (m, 2H), 7.01−6.87 (m, 5H), 5.20 (dd, J = 7.1, 5.6 Hz,
1H), 4.77 (d, J = 7.0 Hz, 1H), 4.05−3.82 (m, 1H), 3.17 (dq, J = 14.0, 7.0
Hz, 1H), 2.39 (s, 3H), 2.14 (s, 3H), 0.99 (t, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, chloroform-d) δ 168.0, 167.3, 161.0 (d, J = 246.8 Hz),
147.0, 139.1, 139.0, 138.6, 133.7, 132.7, 132.5, 132.4, 130.0, 129.9,
129.6 (2C), 128.8, 128.6, 127.7, 125.3, 124.1, 115.5, 115.3, 105.9, 55.7,
+
39.2, 36.7, 21.3, 12.7, 11.9. HRMS (ESI+) m/z calcd for C29H28FN4O2
[M + H]+ 483.2191, found 483.2193.
N-(rel-(4S,5S)-7-Ethyl-4-(4-fluorophenyl)-3-methyl-6-oxo-1-phe-
nyl-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-
(trifluoromethyl)benzamide (Compound 4). Compound 4 was
synthesized by general procedures B−D starting from 4-fluorobenzal-
dehyde, (3-(trifluoromethyl)benzoyl)glycine, and 3-methyl-1-phenyl-
1H-pyrazol-5-amine.
1H NMR (400 MHz, chloroform-d) δ 8.03 (s, 1H), 7.91−7.83 (m,
1H), 7.78 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.54−7.41 (m,
4H), 7.03 (d, J = 5.6 Hz, 1H), 6.96−6.88 (m, 4H), 5.20 (dd, J = 7.3, 5.7
Hz, 1H), 4.76 (d, J = 7.3 Hz, 1H), 3.97 (dq, J = 14.3, 7.2 Hz, 1H), 3.18
(dq, J = 14.0, 7.0 Hz, 1H), 2.15 (s, 3H), 1.00 (t, J = 7.0 Hz, 3H). 13C
NMR (126 MHz, chloroform-d) δ 170.3, 168.4, 165.0 (d, J = 247.0 Hz),
149.7, 141.8, 141.5, 137.3, 135.0 (2C), 134.0 (d, J = 33.2 Hz), 132.7,
132.5, 132.4, 132.3, 132.0, 131.6, 131.2 (q, J = 3.4 Hz), 128.0 (2C),
127.0 (q, J = 3.9 Hz), 126.3 (q, J = 272.4 Hz), 118.3, 118.1, 108.4, 58.5,
42.0, 39.4, 15.4, 14.6. HRMS (ESI+) m/z calcd for C29H25F4N4O2+ [M
+ H]+ 537.1908, found 537.1917.
N-(rel-(4S,5S)-3-(Aminomethyl)-7-ethyl-4-(4-fluorophenyl)-6-
oxo-1-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-
(trifluoromethyl)benzamide (Compound 40). Compound 40 was
synthesized by general procedures A−F starting from 4-fluorobenzal-
dehyde, (3-(trifluoromethyl)benzoyl)glycine, and phenyl hydrazine.
1H NMR (400 MHz, chloroform-d) δ 8.03 (s, 1H), 7.86 (d, J = 7.8
Hz, 1H), 7.80−7.76 (d, J = 7.8 Hz, 1H), 7.61−7.43 (m, 6H), 7.02 (d, J =
5.6 Hz, 1H), 6.99−6.88 (m, 4H), 5.23 (dd, J = 7.3, 5.6 Hz, 1H), 4.88 (d,
J = 7.3 Hz, 1H), 3.97 (dq, J = 14.4, 7.2 Hz, 1H), 3.85−3.70 (m, 2H),
3.19 (dq, J = 13.9, 6.9 Hz, 1H), 1.00 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, chloroform-d) δ 167.7, 165.7, 162.4 (d, J = 247.2 Hz), 151.4,
139.2, 139.1, 134.5, 132.4 (2C), 131.4 (q, J = 32.9 Hz), 130.1, 129.8,
129.7, 129.6, 129.3, 129.1, 128.6 (q, J = 3.7 Hz), 125.4 (2C), 124.3 (q, J
= 3.9 Hz), 122.2 (q, J = 275.4 Hz), 115.8, 115.6, 104.7, 55.8, 39.3, 38.7,
+
36.6, 12.7. HRMS (ESI+) m/z calcd for C29H26F4N5O2 [M + H]+
552.2017, found 552.2028.
N-(rel-(4S,5S)-7-Ethyl-4-(4-fluorophenyl)-6-oxo-1-phenyl-3-(pi-
peridin-1-ylmethyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-b]pyridin-
5-yl)-3-(trifluoromethyl)benzamide (Compound 41). Compound 41
was synthesized by general procedures A−E and H starting from 4-
fluorobenzaldehyde, (3-(trifluoromethyl)benzoyl)glycine, phenyl hy-
drazine, and piperidine.
J. Hydrolysis. To a solution of compound 4 (1.0 mmol) in 1,4-
dioxane (10.0 mL) under nitrogen atmosphere, c-HCl (100 mmol) was
added and refluxed overnight. The mixture was basified with 1 N
NaOH, extracted with DCM, washed with brine, dried with MgSO4,
filtered, and dried. Then, the crude product was purified by flash
chromatography.
1H NMR (400 MHz, chloroform-d) δ 8.03 (s, 1H), 7.85 (d, J = 7.8
Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.60−7.41 (m, 6H), 6.99−6.86 (m,
5H), 5.25−5.17 (m, 1H), 4.96 (d, J = 7.2 Hz, 1H), 3.94 (dq, J = 14.3, 7.2
Hz, 1H), 3.52 (d, J = 13.8 Hz, 1H), 3.31 (d, J = 13.7 Hz, 1H), 3.21 (dq, J
= 14.0, 6.9 Hz, 1H), 2.34−2.10 (m, 4H), 1.33−1.18 (m, 4H), 1.13−
1.04 (m, 2H), 1.01 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz,
K. Amide Coupling. The reaction was performed under either
condition (a) or (b).
(a) To a solution of amine intermediate (1.0 mmol) in CH2Cl2 (1.0
mL), carboxylic acid (1.0 mmol), EDCI·HCl (1.3 mmol), and
DIPEA (1.3 mmol) were added and stirred at room temperature
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chloroform-d) δ 170.7, 168.4, 164.9 (d, J = 246.1 Hz), 151.5, 147.6,
141.9, 141.8, 137.5, 135.8 (q, J = 3.7 Hz), 134.0 (q, J = 33.4 Hz), 132.7,
132.5, 132.4, 132.2, 132.0, 131.7, 131.1 (d, J = 3.8 Hz), 128.4 (q, J =
273.5 Hz), 128.2 (2C), 127.0 (q, J = 3.8 Hz), 118.0, 117.9, 108.9, 58.8,
58.5 (2C), 57.3, 42.1, 39.7, 28.1 (2C), 26.7, 15.4. HRMS (ESI+) m/z
calcd for C34H34F4N5O2+ [M + H]+ 620.2643, found 620.2637.
N-(rel-(4R,5S)-7-Ethyl-4-(4-fluorophenyl)-3-methyl-6-oxo-1-phe-
nyl-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-
(trifluoromethyl)benzamide (Compound 125). Compound 125 was
synthesized by general procedures B−D starting from 4-fluorobenzal-
dehyde, (3-(trifluoromethyl)benzoyl)glycine, and 3-methyl-1-phenyl-
1H-pyrazol-5-amine.
AUTHOR INFORMATION
■
Corresponding Author
R. Kiplin Guy − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40508, United
States; orcid.org/0000-0002-9638-2060; Phone: (859)
257-5290; Email: kip.guy@uky.edu; Fax: (859) 257-2128
Authors
Ho Shin Kim − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40508, United
States
Jared T. Hammill − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40508, United
States
Daniel C. Scott − Department of Structural Biology, St. Jude
Children’s Research Hospital, Memphis, Tennessee 38105,
United States
Yizhe Chen − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40508, United
States
Amy L. Rice − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40508, United
States
William Pistel − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40508, United
States
Bhuvanesh Singh − Department of Surgery, Laboratory of
Epithelial Cancer Biology, Memorial Sloan Kettering Cancer
Center, New York 10065, United States
Brenda A. Schulman − Department of Structural Biology, St.
Jude Children’s Research Hospital, Memphis, Tennessee
38105, United States; Department of Molecular Machines and
Signaling, Max Planck Institute of Biochemistry, Martinsried
82152, Germany
1H NMR (500 MHz, chloroform-d) δ 7.88 (s, 1H), 7.81 (d, J = 7.9
Hz, 1H), 7.73 (d, 1H), 7.57−7.51 (m, 2H), 7.51−7.44 (m, 4H), 7.44−
7.32 (m, 3H), 7.08−7.02 (m, 2H), 6.53 (d, J = 9.0 Hz, 1H), 5.44 (dd, J =
12.8, 9.0 Hz, 1H), 4.12 (d, J = 12.8 Hz, 1H), 3.93 (dq, J = 14.3, 7.1 Hz,
1H), 3.17 (dq, J = 13.9, 6.9 Hz, 1H), 1.60 (s, 3H), 0.92 (t, J = 7.0 Hz,
3H). 13C NMR (101 MHz, chloroform-d) δ 169.5, 166.3, 162.3 (q, J =
247.1 Hz), 146.4, 139.0, 138.3, 134.5, 132.7, 132.7, 131.1 (q, J = 32.8
Hz), 130.5, 130.4, 130.2, 129.5, 129.1, 128.8, 128.3 (q, J = 3.5 Hz),
126.3 (q, J = 273.6 Hz), 125.0 (2C), 124.0 (q, J = 4.0 Hz), 115.8, 115.6,
106.4, 56.3, 42.9, 39.4, 13.0, 12.9. HRMS (ESI+) m/z calcd for
C29H25F4N4O2+ [M + H]+ 537.1908, found 537.1896.
N-(rel-(4S,5S)-3-(Aminomethyl)-7-ethyl-4-(6-fluoropyridin-3-yl)-
6-oxo-1-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-b]pyridin-5-yl)-
3-(trifluoromethyl)benzamide (Compound 143). Compound 143
was synthesized by general procedures A−F starting from 6-
fluoronicotinaldehyde, (3-(trifluoromethyl)benzoyl)glycine, and phe-
nyl hydrazine.
1H NMR (400 MHz, chloroform-d) δ 8.07 (s, 1H), 7.89 (d, J = 7.9
Hz, 1H), 7.87−7.84 (m, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.63−7.45 (m,
6H), 7.39 (td, J = 8.0, 2.5 Hz, 1H), 7.17 (d, J = 4.9 Hz, 1H), 6.81 (dd, J =
8.4, 2.9 Hz, 1H), 5.22 (dd, J = 7.1, 4.9 Hz, 1H), 5.04 (d, J = 7.2 Hz, 1H),
3.98 (dq, J = 14.3, 7.2 Hz, 1H), 3.88−3.73 (m, 2H), 3.17 (dt, J = 13.8,
7.0 Hz, 1H), 0.99 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, chloroform-
d) δ 167.2, 165.8, 163.2 (d, J = 240.3 Hz), 147.5, 147.3, 141.0, 140.9,
139.1, 138.9, 134.1, 131.5 (q, J = 32.8 Hz), 130.0, 129.7, 129.4, 129.3,
128.8 (q, J = 3.1 Hz), 125.5 (2C), 124.4 (q, J = 4.0 Hz), 123.6 (q, J =
273.0 Hz), 109.7, 109.4, 103.6, 55.7, 39.3, 38.7, 34.1, 12.8. HRMS (ESI
+) m/z calcd for C28H25F4N6O2+ [M + H]+ 553.1970, found 553.1967.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00035
Author Contributions
ASSOCIATED CONTENT
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00035.
Funding
R.K.G., B.S., B.A.S., D.C.S., J.T.H., H.S.K., Y.C., A.L.R.: NIH
R01CA247365; B.A.S. and D.C.S.: NIH R37GM069530;
P30CA021765; J.T.H., NIH F32GM113310; ALSAC/St. Jude.
Characterization details for the other synthetic com-
pounds as well as additional information from X-ray
crystallography and biological studies; chemical charac-
terization and purity data; expanded SAR tables including
all compounds; pulse chase data for compound 40;
surface plasmon resonance (SPR) data; metabolite
identification studies for compound 2; in vivo PK data;
data collection and refinement statistics for lysozyme-
DCN1P:1 and Lysozyme-DCN1P:40 (PDF)
Notes
The authors declare the following competing financial
interest(s): Inventors on United States Patent Application
20180256558, METHODS AND COMPOSITIONS OF
INHIBITING DCN1-UBC12 INTERACTION and United
States Patent 10,525,048, Methods and compositions of
inhibiting DCN1-UBC12 interaction. BAS is a member of the
Scientific Advisory Board of Interline Therapeutics.
Molecular formula strings and TR-FRET potency (CSV)
Accession Codes
ACKNOWLEDGMENTS
■
Atomic coordinates and experimental data for the X-ray co-
crystal structures of NAcM-OPT (PDB: 5V86) and 40 (PDB:
7KWA) in complex with lysozyme−DCN1P are available from
the RCSB Protein Data Bank (www.rcsb.org). The authors will
release the atomic coordinates and experimental data upon
article publication.
The authors thank the College of Pharmacy NMR Center
(University of Kentucky) for NMR support. At St. Jude, they
acknowledge the High Throughput Biosciences Center,
Medicinal Chemistry Center, Compound Management, and
High Throughput Analytical Chemistry Centers in Chemical
Biology and Therapeutics, and the Hartwell Center. The authors
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J. Med. Chem. 2021, 64, 5850−5862

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
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thank the staff at the ALS 8.2.1 and Sercat 22-ID beamlines at
the Advanced Light Source and Advanced Photon Source.
ABBREVIATIONS
■
DCN1, defective in cullin neddylation 1; UBL, ubiquitin-like
protein; UBE2M, NEDD8-conjugating enzyme Ubc12;
SCCRO, squamous cell carcinoma-related oncogene; SCC,
squamous cell carcinoma; CRL, cullin-RING ligases; NEDD8,
Neural precursor cell expressed, developmentally downregu-
lated 8; SAR, structure−activity relationship; SPR, structure−
property relationship; TR-FRET, time-resolved fluorescence
resonance energy transfer; HTS, high-throughput screen; t-Bu,
tert-butyl; CETSA, cellular thermal shift assay; ROS, reactive
oxygen species; NAE, NEDD8-activating enzyme; RBX1, Ring-
Box 1, E3 Ubiquitin Protein Ligase; CUL1, cullin 1; AF,
AlexaFluor 488
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J. Med. Chem. 2021, 64, 5850−5862