Route exploration and synthesis of the reported pyridone-based PDI inhibitor STK076545

Article abstract of DOI:10.1039/d0ob01205j

The enzyme protein disulfide isomerase (PDI) is essential for the correct folding of proteins and the activation of certain cell surface receptors, and is a promising target for the treatment of cancer and thrombotic conditions. A previous high-throughput screen identified the commercial compound STK076545 as a promising PDI inhibitor. To confirm its activity and support further biological studies, a resynthesis was pursued of the reported β-keto-amide with an N-alkylated pyridone at the α-position. Numerous conventional approaches were complicated by undesired fragmentations or rearrangements. However, a successful 5-step synthetic route was achieved using an aldol reaction with an α-pyridone allyl ester as a key step. An X-ray crystal structure of the final compound confirmed that the reported structure of STK076545 was achieved, however its lack of PDI activity and inconsistent spectral data suggest that the commercial structure was misassigned.

Full text of DOI:10.1039/d0ob01205j

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Route exploration and synthesis of the reported
pyridone-based PDI inhibitor STK076545†
Cite this: DOI: 10.1039/d0ob01205j
Eric Greve,a Sergey V. Lindeman,a Christina Scartelli,b Lin Lin,b Robert Flaumenhaftb
a
and Chris Dockendorff
*
The enzyme protein disulfide isomerase (PDI) is essential for the correct folding of proteins and the acti-
vation of certain cell surface receptors, and is a promising target for the treatment of cancer and throm-
botic conditions. A previous high-throughput screen identified the commercial compound STK076545 as
a promising PDI inhibitor. To confirm its activity and support further biological studies, a resynthesis was
pursued of the reported β-keto-amide with an N-alkylated pyridone at the α-position. Numerous conven-
tional approaches were complicated by undesired fragmentations or rearrangements. However, a suc-
cessful 5-step synthetic route was achieved using an aldol reaction with an α-pyridone allyl ester as a key
step. An X-ray crystal structure of the final compound confirmed that the reported structure of
STK076545 was achieved, however its lack of PDI activity and inconsistent spectral data suggest that the
commercial structure was misassigned.
Received 11th June 2020,
Accepted 13th August 2020
DOI: 10.1039/d0ob01205j
rsc.li/obc
quercertins, found in high abundance in various fruits and
vegetables, that inhibit PDI. From this class, isoquercertin
Introduction
Protein disulfide isomerase (PDI) is an enzyme primarily loca- (Fig. 1) was found to decrease D-dimer plasma concentrations,
lized in the endoplasmic reticulum that catalyzes the oxi- a biomarker for venous thromboembolic disease, by a median
dation–reduction and isomerization of disulfide bonds and of 22% in a phase II clinical trial.9 However, the high dose and
serves as a necessary chaperone for protein folding.1 In highly variable patients responses are drawbacks of
addition, PDI can be released onto the surface of endothelial isoquercertin.
and platelet cells, where it acts to promote effective coagu-
To seek additional PDI inhibitors, a second high-through-
lation via mechanisms presently under study. For these put screen was performed on 348 505 compounds from the
reasons, PDI inhibitors are of significant interest both for the Molecular Libraries Small Molecule Repository.10 Two series of
treatment of cancer2 and the prevention of thrombosis.3 PDI inhibitors, represented by bepristats 1a and 2a (Fig. 1)
Several animal models of thrombosis have demonstrated that were found to bind to the hydrophobic pocket of the b’
targeting cell surface PDI with antibodies or small molecules
blocks both platelet accumulation and fibrin generation.4–8
Previously reported PDI antagonists suffer from poor selecti-
vity, irreversibility, and/or low potency. In an effort to identify
novel inhibitors of PDI with more suitable therapeutic pro-
perties,
a
high-throughput screen was performed by
Flaumenhaft and co-workers on approximately 5000 bioactive
small molecules.6 They identified a class of flavonoids called
aDepartment of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI,
53201-1881, USA. E-mail: christopher.dockendorff@mu.edu; Tel: +1-414-288-1617
bDivision of Hemostasis and Thrombosis, Department of Medicine, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215,
USA
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra;
additional
X-ray
crystallography
figures
and
discussion.
CCDC
2004020–2004023. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/d0ob01205j
Fig. 1 Select inhibitors of PDI.
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domain.8 A commercial compound called STK076545 (Fig. 1) amine on the amide side chain of STK076045 could compli-
was also identified from the high-throughput screen to be a cate a late stage halogenation/pyridone N-alkylation reaction.
reasonably potent hit for the inhibition of PDI, and had an It remained to be determined how the presence of an
attractive structure for medicinal chemistry studies relative to α-pyridone could affect the steps outlined in Fig. 2. Herein, we
other hits. The commercial supply of STK076545 was soon report our explorations of these routes, culminating in a suc-
depleted, and we were unable to secure additional quantities, cessful 5-step synthesis of the reported structure of
so we immediately endeavored to synthesize it. Its structure STK076545. An initial version of this work was deposited in
proved to be deceptively simple, and this manuscript describes ChemRxiv on May 25th, 2020.21
several pitfalls that were encountered prior to its successful
synthesis.
Methods for preparing β-keto amides have been pursued for
Results and discussion
more than a century.11 The most obvious approach to β-keto
β-Keto carboxylic acid
amides is via amide couplings between β-keto acids and amines,
The initial synthetic route we envisioned to access STK076545
(Fig. 2, approach ‘a’) involved N-alkylation of 2-pyridone with
bromo-β-keto ester 2, followed by ester hydrolysis and amide
coupling (Scheme 1). Using conditions previously reported
which also permits late stage diversification for medicinal chem-
istry studies (Fig. 2, approach ‘a’). However, this approach may
be complicated by the limited stability of the β-keto acid starting
materials, which can undergo decarboxylation (step ‘b’).
Alternatively, Meldrum’s acid can be C-acylated, then aminolysis
affords a β-keto amide, but this approach is limited to
α-unsubstituted substrates.12 Direct aminolysis of β-keto esters13
or β-keto thioesters14 at high temperature is possible (approach
‘c’), but can be compromised by competing enamine formation.
Aminolysis reactions catalyzed with DMAP,15 enzymes,16 or tran-
sition metals17,18 have also been reported. Alternatively, addition
of a ketone or enamine to an isocyanate have also been reported
(approach ‘d’). Cross Claisen-like condensations of esters with
amide enolates have been reported (approach ‘e’),19,20 or alterna-
tively an aldol reaction between a pyridone-containing amide
and a benzaldehyde (Ar = Ph for STK076545) could be envisaged,
followed by alcohol oxidation (step ‘f’).
using
a 3 and
bromomalonate,22 both the N-alkylated
O-alkylated 4 products were isolated, in 47% and 9% yield
respectively. 3 and 4 were readily distinguishable based on
their 13C NMR chemical shifts for the α-carbon, with the
O-alkylated product 4 being assigned based on the more down-
field α-carbon peak at 75.9 ppm. In this paper, all alkylations
of 2-pyridone gave N-alkylation as the major product, though
the O-alkylated products were sometimes observed in trace
amounts. Unfortunately, the hydrolysis of ester 3 under acidic
(H2SO4) or basic conditions (NaOH, LiOH, or Me3SnOH23) all
resulted in decarboxylation of carboxylic acid intermediate 6 to
yield ketone 5, despite careful attempted isolations using
buffered aqueous media. Direct coupling of alkali metal car-
boxylate salts has been shown by Batey and coworkers to be a
useful strategy with unstable carboxylic acids.24 Attempts at a
tandem ester hydrolysis of 3 with NaOH or LiOH followed by
Other approaches involving a late stage addition of the pyri-
done are possible, but these were not initially considered since
we were first interested in exploring amide structure–activity
relationships (SARs), and the presence of a basic tertiary
peptide coupling with carboxylate
7 were not fruitful.
Alternatively, the decarboxylation product 5 was synthesized
on a larger scale via N-alkylation of 2-pyridone with 2-bromoa-
cetophenone. Subsequent carboxylation using MgCl2 and NaI
with CO2 also gave no detectable amount of carboxylate 8 or
carboxylic acid 6 after an acidic workup.25
Fig. 2 Retrosynthetic strategies for accessing the β-keto amide in
STK076545 (Ar = Ph, R1 = –CH2CH2NEt2).
Scheme 1 Ester hydrolysis and carboxylate formation.
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1
via H NMR. Based on our screen, it was found that all reac-
tions occurring at 20 °C facilitated enolate formation (entries
3–5), while no deuterium incorporation occurred when
quenching the samples at −78 °C (entries 1 and 2). The
observed deuterium incorporation over 100% is expected to be
within the systematic error associated with the preparation of
1
LDA or the weight of NaH, and H NMR integrations of these
small scale reactions.
The enolate from ketone 5 was next formed using LDA at
20 °C and reacted with CDI or urea intermediate 16, syn-
thesized
from
CDI
and
N,N-diethylethylenediamine
(Scheme 3). There was no observable reaction with either elec-
trophile, even after heating at 70 °C. We then tested commer-
cially available tert-butyl isocyanate as a model isocyanate for
reaction screening. When LDA was used as a base, no desired
product was observed. Rather, urea byproduct 18 formed from
the addition of diisopropylamine to tert-butyl isocyanate was
detected via LC-MS. Switching to NaH as the base and heating
the reaction at 100 °C for 2 h in toluene yielded amide 19 in
15% yield. Efforts to synthesize the desired isocyanate from
N,N-diethylethylenediamine and triphosgene, or reacting di-
ethylamine with 2-bromoethyl isocyanate, were both trouble-
some. With this synthetic route being low yielding and having
the limitation of only producing secondary amides, we chose
to seek an alternative route.
Scheme 2 Synthesis and deprotection of benzyl ester 13.
In an effort to access carboxylic acid 6 under milder con-
ditions, the analogous benzyl ester intermediate 13 was syn-
thesized in four steps (Scheme 2). First, 1,3-dicarbonyl 10 was
prepared from acetophenone and dimethylcarbonate using
NaH in 98% yield.26 ZnO-catalyzed transesterification of 10
afforded benzyl alcohol 11.27 Next, monohalogenation of 11
with NBS catalyzed by Amberlyst-15®, followed by reaction
with 2-pyridone yielded α-substituted-β-keto ester 13 in 66%
yield over two steps.28 Benzyl removal from ester 13 via palla-
dium-catalyzed hydrogenation also resulted in decarboxyl-
1
ation. In addition, H NMR analysis of the crude product indi-
cated the ketone was reduced to afford benzyl alcohol 14.
Efforts to reduce the ketone prior to ester hydrolysis were not
successful (Scheme 7).
Direct aminolysis of β-keto ester
Another common synthetic approach to access amides is via
direct aminolysis of esters (Fig. 2, approach ‘c’). Starting from
β-keto ester 3, we first tested a Ag(I)-catalyzed aminolysis
(Scheme 4).18 Rather than observing conversion to the desired
β-keto amide, reaction monitoring via LC-MS when using con-
dition A showed masses associated with ester 21 and amide 24
(Scheme 5). Similarly, when heating ester 3 with N,N-diethyl-
ethylenediamine in toluene at 80 °C with or without DMAP,
the same decomposition peaks were present. Ester 21 was iso-
lated when using conditions B and correlated with the mass
peak observed via LC-MS. Ag(I)/DBU and DMAP were found to
Enolate formation from ketone 5
Instead of proceeding through a carboxylic acid intermediate,
we envisioned installation of the amide via reaction of an
enolate with a suitable isocyanate, or reaction with CDI fol-
lowed by addition of an amine to the intermediate acylimida-
zole (Fig. 2, approach ‘d’). To identify suitable conditions for
enolate formation with ketone 5, LDA, LiHMDS, and NaH were
screened as bases (Table 1). Reactions were quenched at
−78 °C or 20 °C using D2O, and crude samples were analyzed
Table 1 Enolate formation from ketone 5
T (°C)
addition
of base
T (°C)
addition
of D2O
Base
(eq.)
% deuterium
Entry Base
incorporationa
1b
2b
3b
4b
5c
LDA
1.05
−78
−78
−78
−78
20
−78
−78
20
20
20
0
0
115
94
134
LiHMDS 1.05
LDA 1.05
LiHMDS 1.05
NaH 1.2
a Deuterium incorporation was determined via 1H NMR. b 12.5 mg of 5
was used in this reaction. c 25 mg of 5 was used in this reaction.
Scheme 3 Enolate reactions with ketone 5.
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Scheme 6 C2–C3 coupling attempts on amide 26a–b.
installation of the C2–C3 bond would occur after amide for-
mation and minimize the possibility of decarboxylation.
Synthesis of 27a-b began with N-alkylation of 2-pyridone with
ethyl bromoacetate followed by ester hydrolysis, affording the
previously reported carboxylic acid 25.30 Amide coupling with
either N,N-diethylethylenediamine 26a or PMB-protected
amine 26b yielded amides 27a-b. We prepared the PMB-pro-
tected amide 26b in an effort to avoid competitive deprotona-
tion of the amide proton during enolization reactions.
Extensive efforts to react these pronucleophiles with different
bases (NaOMe, NaH, and LDA), electrophiles (aldehyde, ester,
or acid chlorides 28a-c), and at variable temperatures
(0–160 °C) were all unfruitful.
Scheme 4 Direct aminolysis attempts with β-keto esters.
Identification of the aldol adduct as a possible intermediate
provided inspiration to access alcohol 32, which would not
undergo decarboxylation during ester hydrolysis (Scheme 7).
Starting from benzyl esters 12 or 13, we attempted to either
protect or reduce the benzylic ketone. Efforts to protect the
ketone using ethylene glycol and catalytic p-TsOH with triethyl
orthoformate and 4 Å mol sieves or a Dean–Stark trap resulted
in no conversion. Alternatively, an attempt to reduce the
ketone with DIBAL-H resulted in pyridone reduction, as
suggested by the crude 1H NMR spectrum. Switching to NaBH4
Scheme 5 Proposed retro Claisen-like condensation decomposition.
both accelerate the conversion to 21 in a few hours, in com-
parison to the reaction heated in toluene that proceeded
slowly over 24 h. We hypothesize that the β-keto ester decom-
poses via a retro Claisen-like condensation mechanism. The
amine (or nucleophilic catalyst) could add to the ketone, fol-
lowed by collapse of the tetrahedral intermediate 23 and clea-
vage of the C–C bond to produce ester 21 (Scheme 5).
Štefane and Polanc reported a method to prepare β-keto
amides from β-keto esters that proceeds via a 1,3,2-dioxabori-
nane intermediate.29 Reacting β-keto ester 3 with boron tri-
fluoride etherate afforded the boron complex 20b in 82% yield
(Scheme 4). Unfortunately, subsequent treatment of 20b with
N,N-diethylethylenediamine also resulted in decomposition to
ester 21 after only 1 h, and complete decomposition after 24 h.
Interestingly, when starting from β-keto ester 10 which does
not have the pyridone substituent in the α position, the prepa-
ration of the boron complex 20a and treatment with N,N-di-
ethylethylenediamine cleanly afforded β-keto amide 22.
Late stage C2–C3 coupling route
Inspired by our observed retro Claisen-like reaction that
occurred with a β-keto ester substrate, we examined the feasi-
bility of performing an aldol addition, Claisen-like conden-
Scheme 7 Attempted protection and reduction reactions of benzylic
sation, or acylation with intermediates 27a-b (Scheme 6). Thus, ketone 12 and 13.
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produced benzyl ester 33, which we presume proceeds via a
similar retro-aldol reaction as observed previously.
Moving forward with the synthesis, methyl ester and acetate
hydrolysis proceeded smoothly to yield the presumed car-
boxylic acid 42a (Scheme 9). At that time, we did not suspect
any issues and completed the synthetic sequence to yield 50,
which was initially thought to be STK076545 (Scheme 10). N,N-
Diethylethylenediamine was used directly for the peptide coup-
ling to prepare 45; however, the subsequent alcohol oxidation
was unsuccessful when using DMP, PDC, IBX, or Bobbitt’s salt
under basic (2,6-lutidine) or acidic (silica gel) conditions.32
Instead, ethanolamine was TBS-protected to afford 44 and
then used in an amide coupling using HATU with carboxylic
acid 42 to yield 46 in 91% yield. DMP oxidation of the alcohol
proceeded smoothly to afford ketone 47, followed by TBS
removal using HCl. In a one-pot reaction, alcohol 48 under-
went a mesylation followed by a substitution with diethyl-
amine. The final product was treated with HCl to furnish the
HCl salt 50 in 28% yield over 2 steps.
Protected benzylic alcohol route
To access benzylic alcohols that could be converted to ketones
late in the synthesis, we prepared bromohydrins 35a-b from
the respective methyl and benzyl cinnamates using NBS and I2
as a catalyst (Scheme 8).31 This also installed alpha halides for
pyridone alkylations. Under standard 2-pyridone alkylation
conditions, epoxide 36 was exclusively formed. To circumvent
this issue, the TBS- or MOM-protected bromohydrins 37a-b
were synthesized. It was found that the use of 2,6-lutidine as
base was critical, as alternative bases such as DIPEA favored
epoxidation over alcohol protection. However, subjecting the
TBS- or MOM-protected bromohydrin to pyridone alkylation
conditions afforded only alkene 38 in <20% yield.
Alternatively, the acetyl-protected alcohol 39 was synthesized
using acetic anhydride and catalytic DMAP. N-Alkylation of
2-pyridone using 39 afforded what was initially presumed to be
the desired product 40a and alkene 41a in 18% and 9% yield
respectively (Scheme 9). Due to the low yields, we screened
alternative solvents (acetone) and bases (Cs2CO3) in an effort
to increase the yield and selectivity; however, the yield of 40a
was not improved.
However, analogs 48 and 50 were both found to be inactive
in a PDI activity assay measuring the reduction of insulin (clea-
vage of its disulfide bonds). Obtained X-ray crystal structures
of 41b and 42b revealed that the pyridone was in the benzylic
position (Scheme 9). A distinct difference between 42a and the
Scheme 8 Preparation of bromohydrin intermediates.
Scheme 10 Synthesis of the incorrect regioisomer of STK076545.
Scheme 9 N-Alkylation of 2-pyridone with acetyl-protected bromohydrin.
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X-ray structure of 42b is the relative stereochemistry between alcohol directly (Scheme 12). Using identical conditions
the hydroxyl and pyridone substituents. We propose that cyclic (LiHMDS and 0 °C), the elimination product 53 was generated.
acetoxonium ion intermediate 43 is formed, similar to that Hydrolysis of ester 53 yielded carboxylic acid 54, which X-ray
proposed for the Prévost33 and Woodward34 dihydroxylation crystallography confirmed to be the Z alkene.
reactions, and 2-pyridone then attacks the benzylic carbon.
With LDA being more commonly used in the literature for
Since we started with (E)-methyl cinnamate, the anti addition aldol addition reactions with esters, we switched our base to
of water to the intermediate rac-bromonium ion results in a LDA (Scheme 12). Quenching the reaction at −78 °C was found
racemic mixture of bromohydrins 39. Formation of the pro- to be critical to avoid generation of the elimination product
posed acetoxonium ion intermediate 43 and subsequent pyri- and give the desired alcohol 55. Unfortunately, ester hydrolysis
done alkyation would result in inversion of both stereocenters with LiOH yielded the elimination product 53 again. In an
and generate 40b. Ester hydrolysis of 40b then yielded acid effort to avoid generation of this undesired alkene, we syn-
42b, with its relative stereochemistry confirmed by the X-ray thesized the TBS-protected alcohol 56. This route was unfruit-
crystal structure (Scheme 9).
ful as ester hydrolysis or cleavage using LiOH, Me3SnOH, or
LiI all resulted in the generation of 53.
Aldol addition route
In order to access the carboxylic acid under milder con-
ditions, we instead synthesized allyl ester 57 (Scheme 13).
First, 2-pyridone was alkylated with allyl chloroacetate to yield
allyl ester 57, and subsequent aldol addition using benz-
aldehyde afforded alcohol 58. By increasing the amount of
benzaldehyde from 1 to 2 equivalents, we were able to nearly
To circumvent the unexpected rearrangement through the
acetoxonium ion intermediate, we planned to perform the
N-alkylation prior to installation of the alcohol/ketone func-
tionality. Inspired by Easton and co-workers’ use of NBS and
AgNO3 to generate hydroxy-α-amino acid derivatives,35 we
sought to access a benzylic bromide intermediate from 51
(Scheme 11). Ester 21 was reacted with LiHMDS to generate an
enolate, followed by alkylation with benzyl bromide to afford
51. Subsequent treatment with NBS and AIBN in MeCN
yielded no bromination at the benzylic position. The m/z peak
observed in the LC-MS trace confirmed the presence of a bro-
1
minated product, but the crude H NMR spectrum suggested
that bromination occurred on the pyridone. Alternatively,
benzaldehyde was used instead of benzyl bromide to react
with the enolate generated from ester 21 to access the benzylic
Scheme 11 Alkylation and attempted benzylic bromination.
Scheme 13 Successful allyl ester route to STK076545.
Scheme 12 Aldol reactions with α-pyridone ester 21.
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supplier (Table 2). This confirms that the structure of the
active PDI-inhibiting compound was misassigned.
Conclusion
Several conventional methods for forming β-keto amides
resulted in fragmentation of pyridone-containing intermedi-
ates, such as retro-Claisen-like and retro-aldol reactions.
Efforts to instead proceed via an acetyl protected bromohydrin
resulted in a rearrangement that we propose proceeds via an
acetoxonium ion intermediate. Alternatively, we successfully
synthesized the reported structure of STK076545 in 5% overall
yield (unoptimized) via a 5-step synthetic route proceeding
through allyl ester 58. This strategy should prove to be broadly
useful in accessing β-keto amides, particularly with an elec-
Fig. 3 X-ray structure of amide 61.
tron-withdrawing
α-substituent
such
as
N-pyridone.
double the yield to 82% for this reaction. Allyl removal with Pd
(PPh3)4 produced carboxylic acid 59. Amide coupling and sub-
sequent DMP oxidation of alcohol 60 successfully produced
the final β-keto-amide product 61 with the reported structure
of STK076545. Incomplete conversion and uncharacterized
byproducts were observed with the low yielding DMP oxi-
dation. Alternative oxidation conditions (e.g. PDC, MnO2,
Bobbitt’s salt,32 and Cu catalyzed aerobic oxidation36) were all
unsuccessful. We obtained an X-ray crystal structure corrobor-
ating the structure of 61 (Fig. 3). Alcohol analog 60, the final
compound 61, and its regioisomeric analog 50 were tested in
an assay measuring PDI-catalyzed insulin reduction and aggre-
gation. All compounds were found to be inactive, with rutin
used as a positive control (Fig. 4, see Experimental section for
details). Interestingly, the 1H NMR spectrum of 61 does not
match that of the STK076545 received from the commercial
Unexpectedly, neither the final compound (61) nor several of
its analogs were found to inhibit protein disulfide isomerase,
and its 1H NMR spectrum does not match that of the active
commercial compound STK076545. It is not uncommon for
complex natural products to have misassigned structures
which require correction after more detailed synthetic and
spectroscopic studies.37 However, it is often taken for granted
that simpler commercial small molecules are provided in high
purity and with structures as advertised, which is not always
the case.38 Our results here highlight the importance of
resynthesis and structure validation of active compounds prior
to embarking on medicinal chemistry campaigns. Current
efforts in our lab are ongoing to elucidate the correct structure
of the commercially supplied compound that may have useful
PDI inhibitory activity.
Experimental section
General information
All reagents and solvents were purchased from commercial
vendors and used as received, except for diethylamine, which
was distilled and stored over 4 Å mol sieves prior to use.
Deionized water was purified by charcoal filtration and used
for reaction workups and in reactions with water. NMR spectra
were recorded on Varian 300 MHz or 400 MHz spectrometers
as indicated. Proton and carbon chemical shifts are reported
in parts per million (ppm; δ) relative to tetramethylsilane,
1
CDCl3, or CD3OD (1H δ 0, 13C δ 77.16, or H δ 3.31, 13C δ 49.0,
Fig. 4 PDI-mediated insulin aggregation assay.
respectively). NMR data are reported as follows: chemical
shifts, multiplicity (br = broad, s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet); coupling constant(s) in Hz;
integration. Unless otherwise indicated, NMR data were col-
lected at 25 °C. NMR data were processed using MestReNova
software. Flash chromatography was performed using Biotage
SNAP cartridges filled with 40–60 µm silica gel, or C18 reverse
phase columns (Biotage® SNAP Ultra 18) on Biotage Isolera
systems, with photodiode array UV detectors. Analytical thin
layer chromatography (TLC) was performed on Agela
Table 2 1H NMR chemical shifts of STK076545 and 61
Entry Compound 1H NMR (CDCl3) δ (integration)
1
STK076545 8.83 (1H), 8.05 (2H), 7.46 (1H), 7.39 (2H), 7.31
(1H), 7.25 (1H), 6.55 (1H), 6.15 (1H), 4.57 (2H),
3.57 (2H), 2.93 (6H), 2.62 (1H), 1.21 (6H)
2
61
8.01 (2H), 7.65 (1H), 7.60–7.56 (1H), 7.48–7.44
(1H), 7.40–7.34 (2H), 6.60 (1H), 6.24 (1H),
3.39–3.28 (2H), 2.57–2.48 (6H), 0.96 (6H)
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Technologies glass plates with 0.25 mm silica gel with F254 turned clear and stopped bubbling (∼10 min). The mixture
indicator. Visualization was accomplished with UV light was diluted with H2O (5 mL) and EtOAc (40 mL) and the layers
(254 nm) and aqueous potassium permanganate (KMnO4) were separated. The organic layer was washed with water (3 ×
stain followed by heating, unless otherwise noted. Tandem 15 mL) and the combined aqueous layers were extracted with
liquid chromatography/mass spectrometry (LC-MS) was per- EtOAc (10 mL). The combined organic layers were washed with
formed on a Shimadzu LCMS-2020 with autosampler, photo- brine (10 mL), dried over anhydrous Na2SO4, filtered, and con-
diode array detector, and single-quadrupole MS with ESI and centrated. The crude yellow solid was dissolved in DCM and
APCI dual ionization, using a Peak Scientific nitrogen genera- purified via flash chromatography (50 g SiO2 column, 0–100%
tor. Unless otherwise noted, a standard LC-MS method was EtOAc/hexanes) to yield pyridone 5 as a white crystalline solid
used to analyze reactions and reaction products: Phenomenex (0.411 g, 77%). This compound has been previously reported
1
Gemini C18 column (100 × 4.6 mm, 3 µm particle size, 110 A and characterized (CAS# 952-75-0). H NMR (CDCl3, 400 MHz)
pore size); column temperature 40 °C; 5 µL of sample in δ 8.02–8.00 (m, 2H), 7.62 (tt, J = 7.4, 1.2 Hz, 1H), 7.51–7.47 (m,
MeOH or CH3CN at a nominal concentration of 1 mg mL−1 1H), 7.40–7.35 (m, 1H), 7.23–7.21 (m, 1H), 6.59 (d, J = 9.2 Hz,
was injected, and peaks were eluted with a gradient of 25–95% 1H), 6.21 (td, J = 6.7, 1.3 Hz, 1H), 2.21 (s, 2H); 13C NMR
CH3CN/H2O (both with 0.1% formic acid) over 5 min, then (CDCl3, 100 MHz) δ 192.4, 162.5, 140.2, 138.4, 134.7, 134.1,
95% CH3CN/H2O for 2 min.
129.0, 128.2, 120.8, 106.1, 54.4.
Methyl 3-oxo-3-phenylpropanoate (10). NaH (3.7 g, 60% in
mineral oil, 92 mmol) and dimethyl carbonate (5.9 g,
Synthetic protocols
Ethyl
3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpro- 66 mmol) were added to a 250 mL oven-dried flask with stir
panoate (3); ethyl 3-oxo-3-phenyl-2-(pyridin-2-yloxy)propanoate bar. A reflux condenser and addition funnel were attached, the
(4). A suspension of 2-hydroxypyridone (163 mg, 1.72 mmol), apparatus was sealed and flushed with N2, and anhydrous
tetrabutylammonium bromide (59.5 mg, 0.184 mmol), and toluene (33 mL) was added under N2. After the mixture was
potassium carbonate (765 mg, 55.3 mmol) in acetone (6.0 mL) heated to reflux, a solution of acetophenone (3.80 mL,
was heated to 40 °C and stirred for 30 min. Ethyl-2-bromo-3- 32.4 mmol) in toluene (17 mL) was added dropwise over 0.5 h.
oxo-3-phenylpropanoate (500 mg, 1.84 mmol) was added and The reaction solution turned orange with the formation of a
the suspension was stirred at 40 °C for 30 min before being white precipitate. After the evolution of hydrogen ceased
cooled to 20 °C. A solution of acetic acid (160 μL, 2.79 mmol) (∼15 min), the reaction was cooled to room temperature.
in water (2.0 mL) was added slowly, and the mixture was Glacial acetic acid (10 mL) was added dropwise and a heavy
stirred for 15 min. The resulting mixture was diluted with H2O pasty solid separated. Ice-cold water was slowly added until the
(2.0 mL) and extracted with DCM (3 × 15 mL). The combined solid dissolved completely, and the reaction mixture was
organic layers were dried over anhydrous Na2SO4, filtered, and diluted with EtOAc (200 mL). The organic layer was separated,
concentrated. The crude orange oil was dissolved in DCM and washed with H2O (200 mL) and brine (200 mL), dried over
purified via flash chromatography (25 g SiO2 column, 0–60% anhydrous Na2SO4, filtered, and concentrated. The residue was
EtOAc/hexanes) to yield pyridone 3 as a yellow oil (229 mg, dissolved in DCM and purified via flash chromatography
47%) and 4 as a yellow oil (44 mg, 9%). 3: Rf = 0.25 (50 : 50 (100 g SiO2 column, 0–25% EtOAc/hexanes) to yield ester 10 as
1
EtOAc : hexanes); H NMR (CDCl3, 400 MHz) δ 8.09 (d, J = 7.3 an orange oil (5.65 g, 98%). This compound has been pre-
Hz, 2H), 7.64–7.58 (m, 2H), 7.51–7.46 (m, 3H), 7.37–7.33 (m, viously reported and characterized (CAS# 614-27-7).26 1H NMR
1H), 6.62 (d, J = 8.7 Hz, 1H), 6.22–6.18 (m, 1H), 4.31–4.28 (m, (300 MHz, CDCl3) δ 12.51 (s, 0.22 H), 7.94 (m, 2H), 7.78 (m,
2H), 1.27 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 0.44 H), 7.76–7.58 (m, 0.44 H), 7.62–7.58 (m, 1 H), 7.50–7.40
191.9, 166.6, 161.4, 140.6, 136.4, 134.8, 134.0, 129.4, 129.2, (m, 2.7 H), 5.68 (s, 0.22 H), 4.01 (s, 2H), 3.80 (s, 0.65 H), 3.75
120.2, 106.4, 62.8, 59.2, 14.1; LC-MS tR = 3.96; m/z = 285.75 (M (s, 3H); 13C NMR (75 MHz, CDCl3) δ 192.5, 173.6, 171.6, 168.0,
+ H); 4: 1H NMR (CDCl3, 400 MHz) δ 8.11–8.05 (m, 3H), 136.0, 133.9, 133.4, 131.4, 128.9, 128.64, 128.6, 126.2, 87.1,
7.63–7.58 (m, 2H), 7.51–7.46 (m, 2H), 6.98–6.92 (m, 2H), 6.68 52.6, 51.5, 45.8.
(s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H); 13C
NMR (CDCl3, 100 MHz) δ 191.7, 166.6, 161.3, 146.5, 139.3, bottom flask was charged with methyl 3-oxo-3-phenylpropano-
Benzyl 3-oxo-3-phenylpropanoate (11). A 25 mL round
133.9, 129.4, 128.7, 118.3, 111.4, 76.0, 62.2, 14.1; LC-MS tR
2.86; m/z = 285.75 (M + H).
=
ate (1.05 g, 5.91 mmol), benzyl alcohol (6.10 mL, 59.1 mmol),
ZnO (96.0 mg, 1.18 mmol) and anhydrous toluene (5 mL). The
1-(2-Oxo-2-phenylethyl)-1,2-dihydropyridin-2-one
(5).
2- flask was fitted with a short-path distillation head and heated
Hydroxypyridine (0.263 g, 2.76 mmol) and Cs2CO3 (1.63 g, in an oil bath set at 110 °C, distilling the methanol formed
5.02 mmol) were added to a 15 mL oven-dried flask with stir during the reaction. After 24 h, LC-MS analysis of the reaction
bar and sealed under N2. Anhydrous DMF (5.0 mL) was added mixture showed complete consumption of the starting
and the suspension was stirred at 20 °C for 30 min before material. The reaction mixture was filtered through a plug of
2-bromoacetophenone (0.500 g, 2.51 mmol) was added and celite and concentrated. The crude residue was dissolved in
the suspension was stirred for an additional 1 h at 20 °C. A DCM and purified via flash chromatography (100 g SiO2
solution of glacial acetic acid (210 μL, 3.77 mmol) in H2O column, 0–20% EtOAc/hexanes) to give benzyl ester 11 as a
(2 mL) was slowly added and the mixture was stirred until it light orange oil (1.43 g, 95%). This compound has been pre-
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viously reported and characterized (CAS# 63888-22-2).39 This This compound has been previously reported and character-
compound exists as a mixture of tautomers in CDCl3 (10 : 1 ized (CAS# 69914-21-2).40 1H NMR (400 MHz, CD3OD) δ
1
keto : enol). H NMR (300 MHz, CDCl3) δ 12.50 (s, enol), 7.94 7.43–7.39 (m, 2H), 7.39–7.33 (m, 3H), 7.29–7.26 (m, 2H), 6.56
(d, J = 8.0 Hz, enol), 7.90 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 7.9 Hz, (d, J = 8.5 Hz, 1H), 6.30 (td, J = 6.7, 1.2 Hz, 1H), 5.01 (dd, J =
enol), 7.59 (t, J = 7.5 Hz, 1H), 7.48–7.28 (m, 7H), 5.73 (s, enol), 8.8, 3.8 Hz, 1H), 4.36 (dd, J = 13.1, 3.8 Hz, 1H), 3.89 (dd, J =
5.25 (s, enol), 5.19 (s, 2H), 4.04 (s, 2H); 13C NMR (75 MHz, 13.1, 8.8 Hz, 1H). 215.75; LC-MS tR = 1.68; m/z = 215.75 (M +
CDCl3) δ 192.4, 167.5, 135.4, 133.9, 128.9, 128.7, 128.6, 128.5, H).
128.4, 128.3, 67.3, 46.1.
N-[2-(Diethylamino)ethyl]-1H-imidazole-1-carboxamide (16).
Benzyl 2-bromo-3-oxo-3-phenylpropanoate (12). In a 100 mL 1,1-Carbonyldiimidazole (205 mg, 1.26 mmol) was added to a
flask with stir bar, ester 11 (1.00 g, 3.93 mmol), 4 mL oven-dried vial with stir bar and sealed under N2.
N-bromosuccinimide (0.735 g, 4.13 mmol), and Amberlyst-15 Anhydrous THF (1.2 mL) was added and the solution was
(2.89 g) in ethyl acetate (30 mL) were stirred at 20 °C for 2.5 h. cooled to 0 °C in an ice bath. A solution of N,N-diethyl-
After completion of the reaction, as indicated by LC-MS, the ethylenediamine (97.9 mg, 0.842 mmol) in anhydrous DCM
reaction mixture was filtered and washed with EtOAc (2 × (0.6 mL) was added dropwise over 10 min and the solution was
20 mL). The combined organic filtrates were dried over anhy- stirred at 20 °C for 1.5 h. Next, the solvent was removed via
drous Na2SO4, and concentrated. The crude product was dis- vacuum and the crude product was loaded on to celite and
solved in DCM and purified via flash chromatography (50 g purified via flash chromatography (12 g C18, MeOH/H2O gradi-
SiO2 column, 0–20% EtOAc/hexanes) to yield alkyl bromide 12 ent) to yield urea 16 as a yellow oil (93.7 mg, 53%). This com-
as a light yellow oil (1.20 g, 92%). This compound has been pound has been previously reported (CAS# 698388-51-1).41 1
H
previously reported and characterized (CAS# 845733-96-2).28 NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 7.45–7.42 (m, 1H),
1H NMR (400 MHz, CDCl3) δ 7.94–7.92 (m, 2H), 7.58 (m, 1H), 7.09–7.07 (m, 2H), 3.46 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 5.6 Hz,
7.43 (m, 2H), 7.30–7.23 (m, 5H) 13C NMR (100 MHz, CDCl3) δ 2H), 2.61–2.51 (m, 4H), 1.06–0.97 (m, 6H); 13C NMR (75 MHz,
188.0, 165.1, 134.6, 134.3, 133.3, 129.2, 128.9, 128.6, 128.6, CDCl3) δ 149.1, 136.1, 130.3, 130.1, 116.1, 51.3, 46.8, 38.2, 11.8;
128.4, 68.8, 46.4.
LC-MS tR = 1.01; m/z = 210.80 (M + H).
Benzyl 3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenyl-pro-
N-tert-Butyl-3-oxo-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenyl
panoate (13). A suspension of 2-hydroxypyridone (212 mg, propanamide (19). In a 4 mL oven-dried vial with a stir bar,
2.23 mmol), tetrabutylammonium bromide (65.4 mg, t-butylisocyanate (7.0 mg, 0.071 mmol) and NaH (60% w/w in
0.203 mmol), and potassium carbonate (0.841 g, 6.09 mmol) mineral oil, 4.7 mg, 0.118 mmol) were mixed in anhydrous
in acetone (4.0 mL) was heated to 40 °C and stirred for 30 min. toluene (0.5 mL) under N2 at 20 °C. Then, ketone 5 (10.0 mg,
Then, bromide 12 (676 mg, 2.03 mmol) in acetone (0.5 mL) 0.047 mmol) was added in one portion and the mixture was
was added. The suspension was stirred at 40 °C for 30 min heated at 100 °C for 2 h. After 2 h, the reaction was quenched
and cooled to room temperature before a solution of acetic by the addition of saturated ammonium chloride. The
acid (232 μL, 4.06 mmol) in water (2.0 mL) was added slowly. aqueous layer was extracted with DCM (3 × 10 mL). The com-
The resulting mixture was stirred for 15 min before it was bined organic extracts were washed with brine (10 mL), dried
diluted with H2O (5.0 mL) and extracted with DCM (3 × over anhydrous Na2SO4, filtered, and condensed to a yellow
15 mL). The combined organic layers were dried over anhy- residue. The crude product was purified via flash chromato-
drous Na2SO4, filtered, and concentrated. The crude dark graphy (10 g SiO2, 0–100% EtOAc/hexanes) to afford amide 19
orange oil was dissolved in DCM and purified via flash chrom- as a yellow oil (2.2 mg, 15%) and recovered ketone 5 (8.0 mg,
atography (50 g SiO2, 0–50% EtOAc/hexanes) to yield pyridone 80%). 1H NMR (300 MHz, CDCl3) δ 7.98 (d, J = 8.0 Hz, 2H),
13 as a colorless oil (496 mg, 70%). 1H NMR (400 MHz, CDCl3) 7.74–7.72 (m, 1H), 7.56–7.52 (m, 1H) 7.43–7.36 (m, 3H), 7.31
δ 8.07–8.04 (m, 2H), 7.64 (s, 1H), 7.60 (tt, J = 7.4, 1.2 Hz, 1H), (s, 1H), 6.85 (br s, 1H), 6.61 (d, J = 9.0 Hz, 1H), 6.29 (t, J = 6.8
7.48–7.44 (m, 3H), 7.35–7.26 (m, 6H), 6.61 (d, J = 9.2 Hz, 1H), Hz, 1H). LC-MS tR = 2.96; m/z = 313.15 (M + H).
6.18 (td, J = 6.8 Hz, 1.3 Hz, 1H), 5.25 (s, 2H). 13C NMR
2,2-Difluoro-6-methoxy-4-phenyl-2H-1λ3,3,2λ4-dioxaborinine
(100 MHz, CDCl3) δ 191.7, 166.5, 161.4, 140.7, 136.4, 134.8, (20a). To an oven-dried 20 mL vial, 10 (0.420 g, 2.36 mmol)
134.6, 133.9, 129.4, 129.2, 128.7, 128.7, 128.4, 120.3, 106.4, and a stir bar were added and the vial was purged with N2 for
68.3, 59.4; LC-MS tR = 3.72; m/z = 348.10 (M + H).
10 min. Then, anhydrous toluene (10 mL) and boron trifluor-
1-(2-Hydroxy-2-phenylethyl)-1,2-dihydropyridin-2-one (14). ide etherate (0.58 mL, 4.71 mmol) were sequentially added.
To a 15 mL flask with stir bar, 13 (33.5 mg, 0.096 mmol) and The reaction mixture was stirred at 20 °C for 20 h. The reaction
MeOH (3.0 mL) were added. The flask headspace was flushed mixture was then concentrated to ∼1/3 of its volume and
with N2 before 10% Pd/C (10.3, 0.0096 mmol) was added. The cooled to −30 °C in a dry ice MeOH/H2O cooling bath. The pre-
flask was flushed with H2 and stirred under H2 (1 atm) at cipitated material was filtered off and washed with 5 : 1 pet-
20 °C for 1 h. After 1 h, the reaction mixture was filtered roleum ether/EtOAc (5 mL), yielding the boron complex 20a as
through celite, condensed under vacuum and analyzed via a yellow solid (307 mg, 58%). 1H NMR (300 MHz, CDCl3) δ
LC-MS and 1H NMR, which indicated conversion to alcohol 14. 7.96–7.92 (m, 2H), 7.63–7.57 (m, 1H), 7.51–7.46 (2H), 6.03 (s,
1H NMR of the crude product showed signals for alkyl protons 1H), 4.12 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 179.6, 176.3,
at 4.36, 3.89, and 5.01 ppm, consistent with published data. 134.0, 132.0, 129.0, 127.9, 83.2, 56.1.
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Ethyl 2-(2-oxo-1,2-dihydropyridin-1-yl)acetate (21). To an was removed under vacuum and 2 M HCl was added to the
oven-dried 1.0 L round bottom flask charged with a stir bar, aqueous solution to reach a pH ∼ 6. Next, the solution was
NaH (60% in mineral oil, 1.99 g, 49.9 mmol) was suspended in concentrated down to dryness and the crude solid was dis-
anhydrous DMF (100 mL) and cooled to 0 °C in an ice bath solved with DCM and purified via flash chromatography (50 g
under N2. To the NaH suspension, a solution of 2-hydroxypyri- SiO2, 0–10% MeOH/DCM w/0.1% formic acid) to afford 25 as a
dine (4.07 g, 42.1 mmol) in anhydrous DMF (200 mL) was white powder (2.04 g, 67%). This compound has been pre-
slowly added. The resulting solution was stirred for 1 h at 0 °C viously reported and characterized (CAS# 56546-36-2).30 1H
before ethyl bromoacetate (4.3 mL, 38 mmol) was added and NMR (400 MHz, acetone-d6) δ 7.62–7.60 (m, 1H), 7.46–7.42 (m,
the mixture was stirred at room temperature for 1.5 h. The 1H), 6.41–6.25 (m, 1H), 6.26–6.21 (m, 1H), 4.71 (s, 2H).
reaction was quenched with saturated NH4Cl (40 mL) and the
[2-(Diethylamino)ethyl][(4-methoxyphenyl)methyl]amine (26b).
product was extracted with DCM (3 × 150 mL). The combined N,N-Diethylethylenediamine (1.25 g, 10.6 mmol) was added to
organic extracts were washed with water (7 × 200 mL), dried a 100 mL oven-dried flask with stir bar and sealed under N2.
over anhydrous Na2SO4, filtered, and concentrated. The crude i-PrOH (25 mL) was added and the flask was cooled to 0 °C.
product was dissolved in DCM and purified via flash chrom- Then, p-anisaldehyde (1.58 mL, 12.8 mmol) was added drop-
atography (100 g SiO2, 0–100% EtOAc/hexanes) to yield ester wise, and the reaction was slowly warmed to 20 °C and stirred
21 as a pale yellow oil (3.62 g, 53%). This compound has been for 16 h. MeOH (20 mL) was added, the solution was cooled to
previously reported and characterized (CAS# 80056-43-5).30 Rf = 0 °C, and NaBH4 (1.69 g, 44.7 mmol) was added in portions
1
0.74 (DCM/MeOH 9 : 1). H NMR (300 MHz, CDCl3) δ 1.29 (t, over 1 h. The solution was stirred for an additional 1 h, while
3H, J = 7.1 Hz), 4.24 (q, 2H, J = 7.1 Hz), 4.64 (s, 2H), 6.18–6.23 warmed to 20 °C. A solution of 10% NaOH in H2O (25 mL) was
(m, 1H), 6.58–6.61 (m, 1H), 7.21–7.24 (m, 1H), 7.34–7.40 (m, added and the resulting mixture was extracted with DCM (3 ×
1H). 13C NMR (100 MHz, CDCl3) δ 14.2, 50.5, 61.9, 106.2, 40 mL). The combined organic layers were washed with a 10%
121.0, 121.0, 138.0, 140.3, 162.5, 167.9.
aqueous solution of NaI (50 mL). The organic layers were dried
N-[2-(Diethylamino)ethyl]-3-oxo-3-phenylpropanamide (22). over Na2SO4, filtered, and concentrated. The resulting crude
N,N-Diethylethylenediamine (167.0 mg, 1.438 mmol) was yellow oil was dissolved in DCM (5 mL) and ether (10 mL),
added to a 15 mL oven-dried flask with stir bar and sealed cooled to 0 °C, and a 4 M HCl solution in 1,4-dioxane (5.6 mL,
under N2. Anhydrous MeCN (5.0 mL) and 20a (250 mg, 22.4 mmol) was added dropwise. The white precipitate was fil-
1.11 mmol) were added, and the reaction mixture was stirred tered and washed with DCM and diethyl ether to yield an off-
at 20 °C for 4 h. After 4 h, an aliquot was removed, condensed white solid. This was dissolved in water (30 mL), cooled to
under reduced pressure, and dissolved in CDCl3 to monitor 0 °C, and the solution was basified with NaOH (to pH ∼ 9).
1
the reaction via H NMR. The reaction mixture was condensed The resulting mixture was extracted with DCM (3 × 40 mL).
under vacuum, dissolved in EtOAc (30 mL), washed with H2O The combined organics were dried over Na2SO4, filtered, and
(2 × 10 mL), dried over MgSO4, filtered, and condensed under concentrated to yield amine 26b as a yellow oil (1.86 g, 74%
vacuum to yield complex 22 as a pale yellow oil (283 mg, 82%). yield). This compound has been previously reported (CAS#
The crude oil (259 mg, 0.834 mmol), sodium acetate (0.342 g, 65875-40-3).42 Rf
=
0.57 (9 : 1 DCM : MeOH); 1H NMR
4.17 mmol), ethanol (5.0 mL), and H2O (5.0 mL) were refluxed (300 MHz, CDCl3) δ 7.23 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz,
for 8 h. TLC analysis (10% MeOH/DCM) of the reaction 2H), 3.79 (s, 3H), 3.74 (s, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.55 (t, J
mixture indicated the starting material was consumed. The = 6.2 Hz, 2H), 2.49 (q, J = 7.1 Hz, 4H), 0.99 (t, 6H); 13C NMR
solvent was removed under reduced pressure, and the residue (75 MHz, CDCl3) δ 158.6, 132.8, 129.3, 113.8, 55.3, 53.5, 52.7,
was dissolved in EtOAc (30 mL) and washed with water (2 × 47.1, 46.9, 11.9; LC-MS tR = 0.89; m/z = 236.90 (M + H).
10 mL). The combined aqueous layers were saturated with
N-[2-(Diethylamino)ethyl]-2-(2-oxo-1,2-dihydropyridin-1-yl)
NaCl and extracted with 9 : 1 DCM : MeOH (5 × 10 mL). The acetamide (27a). Carboxylic acid 25 (37.0 mg, 0.242 mmol)
combined organics were dried over magnesium sulfate, fil- was added to an oven-dried 50 mL flask with stir bar and
tered, and concentrated. The crude oil was purified by flash sealed under N2. To the suspension, anhydrous DCM (4.0 mL),
chromatography (10 g SiO2, 0–10% MeOH/DCM) to yield HOBt (69.4 mg, 0.363 mmol), DIPEA (62.0 μL, 0.362 mmol),
amide 22 as a pale yellow oil (147 mg, 67%). Rf = 0.50 (9 : 1 and N,N-diethyethylenediamine (33.7 mg, 0.290 mmol) were
DCM : MeOH); 1H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 7.4 Hz, sequentially added. The reaction mixture was stirred for 5 min
2H), 7.61–7.57 (m, 1H) 7.50–7.45 (m, 2H), 3.96 (s, 2H), 3.48 (q, at 20 °C before EDC-HCl (69.4 mg, 0.362 mmol) was added in
J = 5.5 Hz, 2H), 2.79–2.73 (m, 6H), 1.14 (t, J = 7.1 Hz, 6H); 13C one portion. The reaction mixture was stirred at 20 °C for 48 h.
NMR (300 MHz, CDCl3) δ 195.16, 178.19, 136.43, 133.82, Monitoring via LC-MS showed conversion to the desired
128.84, 128.70, 51.54, 47.03, 46.53, 36.49, 10.50; LC-MS tR
1.06; m/z = 263.15 (M + H).
=
product. The solvent was removed under vacuum and the
residue was dry loaded using celite and purified via flash
2-(2-Oxo-1,2-dihydropyridin-1-yl)acetic acid (25). Ester 21 chromatography (12 g C18, 0–95% MeOH/H2O gradient w/
(3.59 g, 19.8 mmol) and EtOH : H2O (1 : 1, 60 mL) were added 0.1% NH4OH) to yield amide 27a as a pale yellow oil (21 mg,
1
to a 250 mL oven-dried flask with stir bar and cooled to 0 °C. A 35%). H NMR (300 MHz, CDCl3) δ 7.38–7.38 (m, 2H), 6.61 (d,
1 N aqueous solution of LiOH (40 mL, 40 mmol) was added J = 9.0 Hz, 1H), 6.24 (td, J = 1.3, 6.7 Hz, 1H), 4.56 (s, 2H), 3.30
and the solution was stirred for 2 h at 20 °C. After 2 h, ethanol (q, J = 5.7 Hz, 2H), 2.57–2.50 (m, 7H), 0.99 (t, J = 7.1 Hz, 6H);
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13C NMR (75 MHz, CD3OD) δ 168.1, 163.6, 141.5, 139.8, 139.7, reported
(CAS#
90841-69-3).31
Rf
=
0.38
119.3, 107.2, 52.0, 51.2, 46.9, 36.6, 10.3; LC-MS tR = 0.99; m/z = (70 : 30 hexanes : EtOAc); 1H NMR (300 MHz, CDCl3)
δ
251.80 (M + H).
7.35–7.38 (m, 5H), 5.06 (dd, J = 5.3, 8.4 Hz, 1H), 4.37 (d, J = 8.4
N-[2-(Diethylamino)ethyl]-N-[(4-methoxyphenyl)methyl]-2-(2- Hz, 1H), 3.79 (s, 3H), 3.28 (d, J = 5.3 Hz, 1H); 13C NMR
oxo-1,2-dihydropyridin-1-yl)acetamide (27b). Carboxylic acid (75 MHz, CDCl3) δ 170.0, 139.0, 128.9, 128.7, 127.1, 75.3, 53.3,
25 (0.300 g, 19.6 mmol) was added to an oven dried 50 mL 47.5.
flask with stir bar and sealed under N2. Anhydrous DCE
Benzyl 3-phenyloxirane-2-carboxylate (36). In a 4 mL oven-
(20.0 mL) was added, and the resulting mixture was cooled to dried vial with stir bar, a suspension of 2-hydroxypryidone
0 °C using an ice bath before EDC-HCl (0.563, 29.4 mmol), (6.8 mg, 0.072 mmol), tetrabutylammonium bromide (1.9 mg,
amine 27b (0.300 g, 19.6 mmol), and DMAP (23.9 mg, 0.0060 mmol), and ground potassium carbonate (24.7 mg,
1.96 mmol) were added. The reaction was allowed to warm up 0.179 mmol) in acetone (0.5 mL) was heated at 40 °C for
to 20 °C and stirred under N2 for 16 h. The resulting solution 30 min. A solution of the bromohydrin 35a (20.0 mg,
was washed with 1 M NaOH (10 mL). The aqueous layer was 0.0597 mmol) in acetone (0.2 mL) was added dropwise over
extracted with DCM (2 × 5 mL), and the combined organic 5 min and the mixture was stirred at 40 °C for 30 min. The
layers were dried over anhydrous Na2SO4, filtered, and concen- reaction mixture was monitored via LCMS and TLC (50 : 50
trated. The crude yellow oil was dissolved with DCM and puri- EtOAc : hexanes) and stained with PAA. Saturated NH4Cl
fied via flash chromatography (12 g C18, 0–95% MeOH/H2O (1 mL) and H2O (1 mL) were added the solution was stirred for
with 0.1% NH4OH) to yield amide 27b as a colorless oil 10 min. The resulting solution was diluted with H2O (5 mL)
1
(213 mg, 29%). The H and 13C NMR are complicated due to and extracted with DCM (3 × 10 mL). The combined organic
rotamers. 1H NMR (300 MHz, CDCl3) δ 7.39–7.24 (m, 3H), layers were dried over anhydrous Na2SO4, filtered, and concen-
7.20–7.18 (m, 1H), 6.93–6.91 (m, 1H), 6.85–6.82 (m, 1H), trated. The crude pale yellow oil was dissolved in DCM and
6.58–6.54 (m, 1H), 6.22–6.16 (m, 1H), 4.89 (s, 1H), 4.73 (s, 1H), purified by flash chromatography (5 g SiO2, 0–100% EtOAc/
4.66 (s, 1H), 4.59 (s, 1H), 3.80–3.77 (s, 3H), 3.46–3.37 (m, 2H), hexanes) to yield pyridone 36 as a colorless oil (11.4 mg, 75%).
2.64–2.45 (m, 6H), 1.03–0.95 (m, 6H); 13C NMR (75 MHz, This compound has been previously reported and synthesized
CDCl3) δ 167.0, 166.7, 162.5, 162.4, 159.2, 159.0, 140.0, 140.0, (CAS# 144667-57-2).44 1H NMR (300 MHz, CDCl3) δ 3.55 (d, J =
138.9, 138.7, 129.5, 129.1, 128.2, 127.9, 120.4, 114.3, 114.0, 1.7 Hz, 1H), 4.11 (d, J = 1.7 Hz, 1H), 5.25 (m, 2H), 7.25–7.40
105.8, 105.7, 55.3, 55.2, 51.5, 51.3, 50.3, 49.7, 49.4, 48.9, 47.5, (m, 10H); 13C NMR (75 MHz, CDCl3) δ 168.2, 135.1, 135.0,
47.4, 45.6, 45.3, 45.2, 12.0; LC-MS tR = 1.43; m/z = 371.95 (M + 129.2, 128.8, 128.8, 128.7, 126.0, 67.6, 58.2, 56.9.
H).
Methyl 2-bromo-3-[(tert-butyldimethylsilyl)oxy]-3-phenylpro-
Benzyl 2-bromo-3-hydroxy-3-phenylpropanoate (35a). To a panoate (37a). Bromohydrin 35a (70.0 mg, 0.209 mg) was
25 mL flask with stir bar, benzyl cinnamate (477 mg, added to a 4 mL oven-dried vial with stir bar and sealed under
2.00 mmol), NBS (427 mg, 2.40 mmol) and MeCN : H2O (4 : 1) N2. Anhydrous DCM (1.0 mL), 2.6-lutidine (92.0 μL,
(10 mL) were added and the solution was cooled to 0 °C. 0.794 mmol) and TBSOTf (72.0 μL, 0.314 mmol) were sequen-
Iodine (50.8 mg, 0.200 mmol) was added and the mixture was tially added and the solution was stirred at 20 °C for 2 h. TLC
stirred for 72 h. The reaction mixture was washed with 10% analysis (50 : 50 EtOAc : hexanes) indicated consumption of
aq. sodium thiosulfate (20 mL) and extracted with EtOAc (3 × starting material. The reaction was quenched with the slow
40 mL). The combined organic extracts were washed with addition of saturated NaCl (2 mL), and the mixture was stirred
brine, dried over anhydrous Na2SO4, filtered, and concen- for 15 min. The aqueous layer was extracted with ether (2 ×
trated. The crude product was dissolved in DCM and purified 10 mL), and the organic layer was washed with brine (5 mL),
by flash chromatography (50 g SiO2, 0–100% EtOAc/hexanes) dried over anhydrous Na2SO4, filtered, and condensed. The
to afford bromohydrin 35a as a colorless liquid (276 mg, 41%). crude yellow oil was dissolved in DCM and purified by flash
This compound has been previously reported (CAS# 1332928- chromatography (25 g SiO2, 0–20% EtOAc/hexanes) to yield 37a
1
50-3).43 Rf = 0.47 (50 : 50 EtOAc : hexanes); H NMR (300 MHz, as a pale yellow oil (277 mg, 77%).45 This compound has been
CDCl3) δ 7.23–7.37 (m, 10H), 5.20 (s, 2H), 5.07 (dd, J = 5.5, 8.2 previously reported and synthesized (CAS# 175722-72-2). Rf =
Hz, 1H), 4.41 (d, J = 8.2 Hz, 1H), 3.26 (br d, J = 5.5 Hz, 1H); 13
C
0.68 (70 : 30 hexanes : EtOAc); 1H NMR (300 MHz, CDCl3) δ
NMR (75 MHz, CDCl3) δ 169.3, 139.0, 134.9, 128.9, 128.7, 7.31–7.38 (m, 5H), 4.98 (d, J = 9.8 Hz, 1H), 4.21 (d, J = 9.8 Hz,
128.7, 128.3, 127.0, 75.3, 68.0, 47.8.
1H), 3.81 (s, 3H), 0.79 (s, 9H), 0.01 (s, 3H), −0.29 (s, 3H); 13C
Methyl 2-bromo-3-hydroxy-3-phenylpropanoate (35b). The NMR (75 MHz, CDCl3) δ 169.6, 140.1, 128.7, 127.7, 76.7, 53.0,
procedure for the synthesis of bromohydrin 35a was used with 49.4, 25.6, 18.0, −4.7, −3.3.
the following modifications: methyl trans-cinnamate (1.62 g,
Methyl
2-bromo-3-(methoxymethoxy)-3-phenylpropanoate
10.0 mmol) was used instead of benzyl trans-cinnamate, NBS (37b). Bromohydrin 35a (100 mg, 0.386 mmol) was added to a
(2.14 g, 12.0 mmol), MeCN : H2O (4 : 1) (50 mL), and iodine 4 mL oven-dried vial with stir bar and sealed under N2.
(254 mg, 1.00 mmol). The crude product was dissolved in Anhydrous DCM (1.0 mL) was added and the solution was
DCM and purified by flash chromatography (100 g SiO2, cooled to 0 °C. Then, 2,6-lutidine (67.4 μL, 0.579 mmol) was
0–40% EtOAc/hexanes) to afford bromohydrin 35b as an off- added followed by dropwise addition of methoxychloro-
white solid (1.45 g, 56%). This compound has been previously methane (44 μL, 0.579 mmol). The solution was stirred at 0 °C
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for 1 h and allowed to warm up to room temperature, then 6.66–6.63 (m, 2H), 6.27 (td, J = 6.8, 1.2 Hz, 1H), 3.69 (s, 3H);
stirred at room temperature for 16 h under N2. After 16 h, the 13C NMR (100 MHz, CDCl3) δ 164.0, 161.8, 151.1, 140.6, 137.5,
reaction was diluted with EtOAc (15 mL) and washed with satu- 131.2, 129.5, 129.2, 126.6, 122.1, 115.8, 105.9, 52.0; LC-MS tR =
rated aq. NaHCO3 (10 mL). The aqueous layer was extracted 4.25; m/z = 255.75 (M + H).
with EtOAc (2 × 10 mL), and the combined organic layers were
2-Hydroxy-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoic
dried over anhydrous Na2SO4, filtered, and concentrated. The acid (42b). To a 50 mL flask with stir bar, acetate 40b (1.98 g,
crude yellow oil was dissolved in DCM and purified by flash 6.28 mmol), THF (48 mL), H2O (12 mL), and LiOH–H2O
chromatography (5 g SiO2, 0–40% EtOAc/hexanes) to yield 37b (580 mg, 13.8 mmol) were sequentially added and the reaction
as
a
colorless oil (78 mg, 66%). Rf
=
0.59 was stirred at 20 °C for 30 min. Analysis via LC-MS indicated
complete conversion. THF was removed under vacuum before
(70 : 30 hexanes : EtOAc); 1H NMR (300 MHz, CDCl3)
δ
7.40–7.35 (m, 5H), 5.00 (d, J = 10.1 Hz, 1H), 4.54–4.45 (m, 2H), a 1 M HCl solution was added dropwise until the pH reached
4.33 (d, J = 10.1 Hz, 1H), 3.85 (s, 3H), 3.25 (s, 3H); 13C NMR ∼1. The solution was concentrated, the crude product was dry
(75 MHz, CDCl3) δ 169.4, 136.9, 129.1, 128.7, 128.6, 128.4, loaded using celite, and purified via flash chromatography
128.4, 127.1, 94.8, 78.7, 56.1, 53.1, 47.3.
Methyl 3-(acetyloxy)-2-bromo-3-phenylpropanoate
(12 g C18, 0–95% 0.5 N NH3 in MeOH/H2O) to afford car-
(39). boxylic acid 42b as an off-white solid (1.49 g, 92%). H NMR
1
Bromohydrin 35b (3.28 g, 12.6 mmol) was added to a 50 mL (400 MHz, DMSO-d6) δ 7.49–7.47 (m, 3H), 7.37–7.29 (m, 4H),
oven-dried flask with stir bar and sealed under N2. Anhydrous 6.43–6.40 (m, 2H), 6.15 (t, J = 6.7 Hz, 1H), 4.66 (d, J = 5.0 Hz,
DCM (20.0 mL), acetic anhydride (1.34 mL, 14.3 mmol) and 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.2, 161.2, 139.7,
DMAP (61.8 mg, 0.506 mmol) were sequentially added. The 137.6, 136.8, 129.3, 128.6, 128.1, 119.2, 105.0, 70.4, 58.2;
solution was stirred at 20 °C for 16 h. TLC analysis LC-MS tR = 3.34; m/z = 258.05 (M − H).
(50 : 50 hexanes : EtOAc) confirmed consumption of starting
(2-Aminoethoxy)(tert-butyl)dimethylsilane
(44).
material. The reaction mixture was poured into ice cold H2O Ethanolamine (2.07 g, 33.9 mmol) was added to a 25 mL oven-
(100 mL), and extracted with DCM (3 × 100 mL). The combined dried flask with stir bar and sealed under N2. Imidazole
organic layers were washed with brine (100 mL), dried over (4.61 g, 67.8 mmol) and anhydrous DCM (30 mL) were added
anhydrous Na2SO4, filtered, and concentrated. The crude pale before a solution of TBSCl (5.11 g, 33.9 mmol) in anhydrous
yellow oil was dissolved in DCM and purified by flash chrom- DCM (5 mL) was added dropwise over 10 min via syringe
atography (100 g SiO2, 0–20% EtOAc/hexanes) to yield 39 as a pump at 20 °C. The solution was allowed to stir at room temp-
colorless oil (3.55 g, 93%). This compound has been previously erature for 1 h. Next, the solution was diluted with DCM
reported and synthesized (CAS# 59339-56-9).46 Rf = 0.73 (50 : 50 (150 mL) and washed with water (3 × 50 mL) and brine
EtOAc : hexanes); 1H NMR (300 MHz, CDCl3) δ 7.43–7.35 (m, (50 mL). The organic layer was dried over MgSO4, filtered, and
5H), 6.11 (d, J = 9.9 Hz, 1H), 4.50 (d, J = 9.9 Hz, 1H), 3.81 (s, concentrated to give amine 44 as a pale yellow oil (4.77 g,
3H), 2.02 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 168.8, 168.3, 80%). This compound has been previously reported and syn-
136.0, 129.3, 128.6, 128.0, 75.6, 53.3, 46.1, 20.9.
thesized (CAS# 101711-55-1).47 1H NMR (CDCl3, 400 MHz) δ
Methyl 2-(acetyloxy)-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phe- 3.63 (t, J = 5.2 Hz, 2H), 2.77 (t, J = 5.2 Hz, 2H), 1.47, (br s, 2H),
nylpropanoate (40b). 2-Hydroxypyridine (3.55 g, 37.4 mmol) 0.91 (s, 9H), 0.07 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 65.5,
and Cs2CO3 (8.11 g, 24.9 mmol) were added to a 250 mL oven- 44.5, 26.1, 18.5, −5.2.
dried flask with stir bar and sealed under N2. Anhydrous DMF
N-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-2-hydroxy-3-(2-oxo-1,2-
(75 mL) was added and the suspension was heated at 50 °C for dihydropyridin-1-yl)-3-phenylpropanamide (46). To an oven-
1 h, then cooled to 20 °C. A solution of alkyl bromide 39 dried 50 mL round bottom flask, acid 42 (420 mg, 1.62 mmol),
(7.50 g, 24.9 mmol) in anhydrous DMF (20 mL) was added and amine 44 (568 mg, 3.24), anhydrous DMF (10 mL), and anhy-
the reaction mixture was stirred at 20 °C for 16 h. The reaction drous DCE (10 mL) were added. Next, NMM (356 μL,
was quenched with saturated aq. NH4Cl (75 mL), diluted with 3.24 mmol) was added via syringe and HATU (739 mg,
EtOAc (500 mL), and washed with H2O (6 × 200 mL). The 1.94 mmol) was added in one portion. The reaction mixture
organic layer was dried over anhydrous Na2SO4, filtered, and was stirred at 20 °C for 16 h under N2. After 16 h, the reaction
concentrated. The resulting crude yellow oil was dissolved in mixture was diluted with EtOAc (200 mL) and washed with H2O
DCM and purified by flash chromatography (100 g SiO2, (2 × 50 mL) and brine (50 mL). The organic layer was dried over
0–100% EtOAc/hexanes) to yield 40b as a waxy off-white solid anhydrous Na2SO4, filtered, and condensed under vacuum to
(1.98 g, 25%) and 41b as an off-white solid (0.60 g, 9%). 40b: yield a crude yellow oil. The crude oil was purified by flash
1H NMR (400 MHz, CDCl3) δ 7.49–7.47 (m, 2H), 7.39–7.38 (m, chromatography (25 g SiO2, 0–100% EtOAc/hexanes) to afford
1
3H), 7.27–7.25 (m, 1H), 7.05 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 5.1 amide 46 as a colorless oil (620 mg, 91%). H NMR (400 MHz,
Hz, 1H), 6.60 (d, J = 9.7 Hz, 1H), 6.02 (t, J = 6.7 Hz, 1H), 5.89 CDCl3) δ 7.44–7.40 (m, 1H), 7.35–7.30 (m, 7H), 6.81 (br s, 1H),
(d, J = 7.1 Hz, 1H), 3.65 (s, 3H), 2.14 (s, 3H); 13C NMR 6.68 (d, J = 9.1 Hz, 1H), 6.25 (t, J = 7.3 Hz, 1H), 6.00 (s, 1H),
(100 MHz, CDCl3) δ 169.4, 167.9, 162.2, 139.4, 135.9, 134.6, 4.84 (s, 1H), 3.66–3.53 (m, 2H), 3.42–3.30 (m, 2H), 0.87 (s, 9H),
129.5, 129.3, 129.1, 120.7, 105.8, 71.3, 57.2, 53.0, 20.7; LC-MS 0.02 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 170.9, 164.9, 140.6,
tR = 5.50; m/z = 255.75 (M + H). 41b: 1H NMR (400 MHz, 139.2, 135.0, 128.7, 128.6, 128.4, 121.6, 107.7, 74.5, 71.5, 61.8,
CDCl3) δ 7.47–7.40 (m, 7H), 7.10 (ddd, J = 6.8, 2.1, 0.7 Hz, 1H), 41.6, 26.0, 18.4, −5.3; LC-MS tR = 5.74; m/z = 417.00 (M + H).
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N-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-2-oxo-3-(2-oxo-1,2-
Ethyl 2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoate
dihydropyridin-1-yl)-3-phenylpropanamide (47). Alcohol 46 (51). In a 20 mL oven-dried vial with stir bar, HMDS (1.90 mL,
(205 mg, 0.492 mmol) was added to a 20 mL oven-dried vial 9.11 mmol) and anhydrous THF (9.0 mL) were added under
with stir bar and sealed under N2. Anhydrous DCM (10 mL) N2. The solution was cooled to 0 °C in an ice bath before
and DMP (251 mg, 0.592 mmol) were added and the reaction n-butyl lithium (5.43 mL of a 1.6 M solution in hexanes,
was stirred at 20 °C for 2 h under N2. A 10% Na2S2O3 aqueous 8.69 mmol) was added dropwise over 10 min. The solution was
solution (10 mL) was added and the biphasic mixture was cooled to −78 °C before a solution of ester 21 (1.50 g,
stirred for 20 min until the two layers became clear. The 8.28 mmol) in anhydrous THF (25.0 mL) was added dropwise.
aqueous layer was separated and the organic layer was washed The solution was stirred at −78 °C for 1 h under N2 before ben-
with saturated NaHCO3 (2 × 5 mL). The combined aqueous zylbromide (985 μL, 8.28 mmol) was added dropwise. The reac-
layers were extracted with EtOAc (1 × 10 mL) and the combined tion mixture was stirred at −78 °C for 1 h, allowed to warm up
organics were dried over anhydrous Na2SO4, filtered, and con- to 0 °C, quenched with saturated NH4Cl (10 mL), and diluted
centrated. The crude oil was dissolved in DCM and purified with EtOAc (50 mL). The organic layer was separated, washed
via flash chromatography (25 g SiO2, 0–80% EtOAc/hexanes) to with brine, dried over anhydrous Na2SO4, filtered, and concen-
1
afford ketone 47 as an off-white solid (170 mg, 83%). H NMR trated. The crude material was dissolved in DCM and purified
(400 MHz, CDCl3) δ 7.38–7.26 (m, 7H), 6.93 (d, J = 7.0 Hz, 1H), via flash chromatography (100 g SiO2, 0–40% EtOAc/hexanes)
6.85 (s, 1H), 6.52 (d, J = 9.1 Hz, 1H), 6.08 (t, J = 6.7 Hz, 1H), to afford 51 as a colorless oil (1.52 g, 68%). 1H NMR (300 MHz,
3.64–3.60 (m, 2H), 3.43–3.25 (m, 2H), 0.80 (s, 9H), −0.04 (s, CDCl3) δ 7.28–7.18 (m, 4H), 7.11–7.08 (m, 2H), 7.03 (dd, J =
6H); 13C NMR (100 MHz, CDCl3) δ 189.8, 162.4, 159.4, 140.2, 6.9, 1.5 Hz, 1H), 6.50 (d, J = 9.2 Hz, 1H), 6.03 (td, J = 6.8, 1.3
135.5, 130.7, 130.2, 130.0, 129.7, 119.7, 106.5, 64.3, 61.3, 41.5, Hz, 1H), 5.41 (dd, J = 9.7, 5.6 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H),
25.9, 18.2, −5.4; LC-MS tR = 6.09; m/z = 414.95 (M + H).
N-(2-Hydroxyethyl)-2-oxo-3-(2-oxo-1,2-dihydropyridin-1-yl)-3- (75 MHz, CDCl3) δ 169.5, 162.2, 139.6, 136.5, 136.1, 129.1,
3.50 (m, 1H), 3.33 (m, 1H), 1.23 (t, J = 7.1 Hz, 3H); 13C NMR
phenylpropanamide (48). To a 100 mL flask charged with a 128.7, 127.1, 120.8, 105.6, 61.9, 61.2, 36.3, 14.1; LC-MS tR
stir bar, silyl ether 47 (100 mg, 0.241 mmol) and MeOH 4.67; m/z = 271.90 (M + H).
=
(10.0 mL) were added. Then a 2% aqueous HCl in MeOH solu-
Ethyl (2Z)-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylprop-2-
tion (10.0 mL) was added, and the reaction was stirred for 1 h enoate (53). The procedure for the synthesis of 51 was followed
at 20 °C. The solution was concentrated, dry loaded on to using HMDS (392 mg, 2.43 mmol), anhydrous THF (9.0 mL),
celite, and purified via flash chromatography (12 g C18, 0–40% n-butyl lithium (1.45 mL of a 1.6 M solution in hexanes,
MeOH/H2O) to afford alcohol 48 as a colorless oil (56.7 mg, 2.32 mmol), 21 (400 mg, 2.21 mmol) in anhydrous THF
78%). LC-MS tR = 3.64; m/z = 300.85 (M + H). This intermediate (3.0 mL), and benzaldehyde (0.247 mL, 2.43 mmol) instead of
was used directly in the next step.
benzylbromide. After workup, the product was dissolved in
Diethyl({2-[2-oxo-3-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenyl- DCM and purified via flash chromatography (25 g SiO2, 0–50%
propanamido]ethyl})azanium chloride (50). Alcohol 48 EtOAc/hexanes) to afford alkene 53 as an off-white solid
1
(40.0 mg, 0.133 mmol) was added to a 20 mL oven-dried vial (272 mg, 46%). H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.43
with stir bar and sealed under Ar. Anhydrous MeCN (4.0 mL), (ddd, J = 9.0, 6.6, 2.0 Hz, 1H), 7.33–7.26 (m, 3H), 7.20–7.18 (m,
DIPEA (22.8 μL, 0.133 mmol), and MsCl (30.9 μL, 0.400 mmol) 2H), 6.96 (dd, J = 6.7, 1.8 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.18
were respectively added and the solution was stirred at 20 °C (td, J = 6.7, 1.0 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1
for 24 h. Analysis via TLC confirmed conversion to the mesy- Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 163.4, 162.4, 140.8,
late. Diethylamine (275 μL, 2.66 mmol) was added and the 137.7, 137.4, 131.4, 130.5, 130.2, 129.9, 128.9, 121.8, 106.8,
reaction was heated at 70 °C for 16 h under Ar. The reaction 62.0, 14.1. LC-MS tR = 6.06; m/z = 269.80 (M + H).
was concentrated, dry loaded using celite, and purified via
(2Z)-2-(2-Oxo-1,2-dihydropyridin-1-yl)-3-phenylprop-2-enoic
flash chromatography (10 g C18, MeOH/H2O gradient w/0.1% acid (54). To a 15 mL flask with stir bar, ester 53 (136 mg,
formic acid) to afford the free base of amine 50 as an off-white 0.504 mmol), THF (6.0 mL), H2O (1.5 mL), and LiOH–H2O
powder (11.0 mg, 28%). Rf = 0.61 (95 : 5 DCM : MeOH); 1H (25.4 mg, 0.604 mmol) were added and the solution was
NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 8.03 (s, 1H), 7.51–7.49 stirred at 20 °C for 12 h. THF was removed under vacuum
(m, 3H), 7.41 (ddd, J = 8.8, 6.6, 1.9 Hz, 1H), 7.37–7.35 (m, 2H), before a 1 M HCl solution was added dropwise until the pH
6.90 (dd, J = 7.1, 1.7 Hz, 1H), 6.55 (d, J = 8.8 Hz, 1H), 6.15 (td, J reached ∼1. The solution was concentrated under vacuum and
= 6.6, 1.2 Hz, 1H), 6.07 (s, 1H), 4.73–4.70 (m, 1H), 4.26–4.22 the crude product was dry loaded using celite and purified via
(m, 1H), 3.86–3.58 (m, 6H), 1.36–1.24 (m, 6H). This compound flash chromatography (12 g C18, 0–75% MeOH/H2O w/0.1%
had poor solubility in chloroform and MeOH. The HCl salt formic acid) to afford carboxylic acid 54 as an off-white solid
was formed prior to 13C NMR analysis. The product was taken (108 mg, 89%). 1H NMR (400 MHz, CD3OD) δ 7.94 (s, 1H), 7.65
up into minimal H2O and 0.5 mL of 1 M HCl was added. The (ddd, J = 9.1, 6.7, 2.0 Hz, 1H), 7.39–7.30 (m, 4H), 7.20–7.18 (m,
solution was lyophilized to afford a yellow oil. 13C NMR 2H), 6.67 (d, J = 9.1 Hz, 1H), 6.42 (td, J = 6.7, 1.0 Hz, 1H); 13C
(75 MHz, D2O) δ 170.4, 164.2, 155.1, 143.0, 136.5, 131.0, 131.0, NMR (100 MHz, CD3OD) δ 166.2, 164.8, 143.6, 140.0, 139.0,
130.4, 129.9, 129.8, 118.9, 109.2, 64.8, 64.6, 49.7, 45.5, 41.9, 133.1, 131.7, 131.6, 130.9, 130.1, 121.6, 109.4; LC-MS tR = 4.17;
13.3, 10.6; LC-MS tR = 1.61; m/z = 356.40 (M + H).
m/z = 241.75 (M + H).
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Ethyl 3-hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenyl- chloroacetate (5.98 mL, 50.5 mmol) was added and the
propanoate (55). In a 100 mL oven-dried flask with stir bar, mixture was stirred at 20 °C for 12 h. The reaction was
diisopropylamine (309 μL, 2.21 mmol) and anhydrous THF quenched with saturated NH4Cl (200 mL) and diluted with
(35.0 mL) were added via syringe under N2. The solution was EtOAc (750 mL). The organic layer was washed with water (6 ×
cooled to −78 °C in a dry ice/acetone bath before n-butyl 200 mL), dried over anhydrous Na2SO4, filtered, and concen-
lithium (1.38 mL of a 1.6 M solution in hexanes, 2.21 mmol) trated. The crude product was dissolved in DCM and purified
was added dropwise over 10 min. The solution was allowed to via flash chromatography (100 g SiO2, 0–85% EtOAc/hexanes)
stir for 30 min before a solution of 21 (400 mg, 2.21 mmol) in to afford pyridone 57 as a pale yellow oil (3.40 g, 42%). 1H
anhydrous THF (3.0 mL) was added dropwise over 5 min. The NMR (400 MHz, CDCl3) δ 7.30 (ddd, J = 9.2, 6.7, 2.1 Hz, 1H),
heterogenous solution was stirred at −78 °C for 1 h before 7.20 (ddd, J = 6.7, 2.1, 0.7 Hz, 1H), 6.49 (ddd, J = 9.2, 1.4, 0.7
benzaldehyde (0.247 mL, 2.43 mmol) was added, and the solu- Hz, 1H), 6.13 (td, J = 6.7, 1.4 Hz, 1H), 5.82 (ddt, J = 17.2, 10.5,
tion was stirred at −78 °C for 2 h. The reaction was quenched 5.7 Hz, 1H), 5.25 (dq, J = 17.2 1.5 Hz, 1H), 5.17 (dq, J = 10.5,
at −78 °C via the addition saturated aq. NH4Cl (15 mL) and 1.3 Hz, 1H), 4.60 (s, 2H), 4.58 (dt, J = 5.7, 1.4 Hz, 2H). 13C NMR
diluted with EtOAc (75 mL). The organic layer was separated, (100 MHz, CDCl3) δ 167.5, 162.4, 140.3, 138.0, 131.3, 121.0,
washed with brine, dried over anhydrous Na2SO4, filtered, and 119.0, 106.2, 66.3, 50.5. LC-MS tR = 2.30; m/z = 181.90 (M + H).
concentrated. The crude material was dissolved in DCM and
Prop-2-en-1-yl 3-hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-
purified via flash chromatography (50 g SiO2, 0–100% EtOAc/ phenylpropanoate (58). In an oven-dried 100 mL flask with stir
hexanes) to afford alcohol 55 as a colorless oil (307 mg, 48%). bar, anhydrous THF (50.0 mL) and diisopropylamine (1.55 mL,
1
The NMR spectra are complex due to diastereomers. H NMR 11.0 mmol) were added via syringe under N2. The solution was
(300 MHz, CDCl3) δ 7.59 (dd, J = 6.8, 1.3 Hz, 1H), 7.26–7.15 (m, cooled to −78 °C before n-butyl lithium (6.90 mL of a 1.6 M
12H), 6.80 (dd, J = 6.8, 1.5 Hz, 1H), 6.39 (d, J = 9.1 Hz, 1H), solution in hexanes, 11.0 mmol) was added dropwise over
6.28 (d, J = 9.1 Hz, 1H), 6.05 (td, J = 6.7, 1.2 Hz, 1H), 5.86 (td, J 5 min. The solution was stirred for 30 min before a solution of
= 6.7 Hz, 1.2 Hz, 1H), 5.81 (d, J = 4.3 Hz, 1H), 5.63 (d, J = 4.3 ester 57 (1.94 g, 10.0 mmol) in anhydrous THF (8.0 mL) was
Hz, 1H), 5.45 (d, J = 7.5 Hz, 1H), 4.74 (d, J = 7.5 Hz, 1H), added dropwise over 5 min and stirred at −78 °C for 1 h.
4.22–4.16 (m, 4H), 1.24–1.19 (m, 6H); 13C NMR (75 MHz, Benzaldehyde (1.53 mL, 15.1 mmol) was added and the result-
CDCl3) δ 169.4, 168.2, 162.9, 162.3, 140.2, 140.1, 139.5, 138.9, ing solution was stirred at −78 °C for 2 h. The reaction was
138.5, 137.9, 128.4, 128.3, 127.8, 126.4, 125.9, 120.3, 119.7, quenched at −78 °C with saturated aq. NH4Cl and diluted with
105.9, 105.6, 73.5, 72.1, 67.5, 63.5, 62.1, 62.1, 60.4, 21.1, 14.2, EtOAc (200 mL) and H2O (10 mL). The layers were
14.1, 14.0; LC-MS tR = 4.78, 4.92; m/z = 287.80 (M + H).
separated and the organic layer was washed with brine, dried
Ethyl 3-[(tert-butyldimethylsilyl)oxy]-2-(2-oxo-1,2-dihydropyr- over anhydrous Na2SO4, and concentrated. The crude yellow
idin-1-yl)-3-phenylpropanoate (56). The procedure for the syn- oil was dissolved in DCM and purified via flash chromato-
thesis of 37a was used with the following modifications: 55 graphy (100 g SiO2, 0–70% EtOAc/hexanes) to afford alcohol 58
1
(153 mg, 0.533 mmol), DCM (6.0 mL), 2,6-lutidine (74.4 μL, as a yellow oil (2.45 g, 82%). H NMR (400 MHz, CDCl3) δ 7.43
0.639 mmol) and TBSOTf (147 μL, 0.639 mmol) were used. The (d, J = 7.9 Hz, 1H), 7.27–7.22 (m, 13H), 6.71 (dd, J = 6.8, 1.5 Hz,
crude oil was dissolved in DCM and purified via flash chrom- 1H), 6.46 (d, J = 9.1 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 6.09 (td, J
atography (25 g SiO2, 0–50% EtOAc/hexanes) to yield 56 as a = 6.8, 1.3 Hz, 1H), 5.94–5.81 (m, 3H), 5.73 (s, 2H), 5.57 (d, J =
pale yellow oil (187 mg, 87%). 1H NMR (300 MHz, CDCl3) δ 7.9 Hz, 1H), 5.34–5.20 (m, 4H), 4.72–4.64 (m, 6H); 13C NMR
8.20 (dd, J = 6.9, 1.4 Hz, 1H), 7.87 (dd, J = 6.9, 1.4 Hz, 1H), (100 MHz, CDCl3) δ 169.2, 167.9, 163.1, 162.4, 140.3, 140.2,
7.56–7.38 (m, 11H), 6.62 (d, J = 9.2 Hz, 1H), 6.53 (d, J = 9.2 Hz, 139.4, 138.7, 138.5, 137.9, 131.4, 131.3, 128.5, 128.4, 128.4,
1H), 6.44–6.39 (m, 1H), 6.33–6.28 (m, 1H), 6.12 (d, J = 7.2 Hz, 128.0, 126.4, 125.9, 120.5, 120.0, 119.1, 119.0, 106.0, 105.8,
1H), 5.87 (d, J = 3.9 Hz, 1H), 5.44 (d, J = 7.2 Hz, 1H), 4.59–4.51 73.7, 72.1, 68.0, 66.6, 66.6, 64.0; LC-MS tR = 4.40, 4.53; m/z =
(m, 1H), 4.45–4.33 (m, 3H), 1.53 (t, J = 7.1 Hz, 3H), 1.46 (t, J = 299.75 (M + H).
7.1 Hz, 3H), 1.13 (s, 9H), 1.11 (s, 9H), 0.29 (s, 3H), 0.25 (s, 3H),
3-Hydroxy-2-(2-oxo-1,2-dihydropyridin-1-yl)-3-phenylpropanoic
0.02 (s, 3H), 0.00 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.0, acid (59). Alcohol 58 (1.90 g, 0.264 mmol) and Pd(PPh3)4
168.5, 162.2, 161.8, 139.6, 139.5, 139.4, 139.3, 138.7, 136.6, (12.2 mg, 0.0106 mmol) were added to a 500 mL oven-dried
128.3, 128.2, 128.2, 128.2, 127.0, 126.4, 120.4, 119.7, 105.4, flash with stir bar and sealed under N2. Anhydrous THF
104.5, 75.6, 75.4, 62.1, 61.9, 61.7, 61.5, 25.8, 25.8, 18.1, 18.1, (6.0 mL) and morpholine (24.2 μL, 0.277 mmol) were added
14.2, 14.1, −4.4, −4.6, −5.3, −5.5; LC-MS tR = 6.95; m/z = 401.95 via syringe and the reaction was stirred at 20 °C for 30 min.
(M + H).
After 30 min, analysis via LC-MS indicated consumption of
Prop-2-en-1-yl 2-(2-oxo-1,2-dihydropyridin-1-yl)acetate (57). starting material. The reaction mixture was concentrated, dry
NaH (60% dispersion in mineral oil, 1.77 g, 46.3 mmol) was loaded using celite, and purified using flash chromatography
added to a 500 mL oven-dried flask with stir bar and sealed (30 g C18, 0–95% MeOH/H2O w/0.1% formic acid) to afford
under N2. Anhydrous DMF (125 mL) was added and the sus- carboxylic acid 59 as a yellow oil (1.20 g, 73%). 1H NMR
pension was cooled to 0 °C in an ice bath. A solution of (400 MHz, CD3OD) δ 8.10 (d, J = 6.5 Hz, 1H), 7.44 (d, J = 6.5
2-hydroxypyridine (4.00 g, 42.1 mmol) in anhydrous DMF Hz, 1H), 7.36–7.15 (m, 10H), 6.37 (d, J = 9.0 Hz, 1H), 6.30–6.27
(25.0 mL) was slowly added and stirred for 1 h at 0 °C. Allyl (m, 1H), 6.14–6.10 (m, 1H), 5.73 (d, J = 3.8 Hz, 1H), 5.45 (d, J =
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7.9 Hz, 1H), 5.30 (d, J = 7.9 Hz, 1H). LC-MS tR = 3.74, 3.88; m/z for 1 h. For calculation of the IC50, we used absorbance values
= 259.75 (M + H). recorded at 25 min, after insulin aggregation is first detectable
N-[2-(Diethylamino)ethyl]-3-hydroxy-2-(2-oxo-1,2-dihydropyri- in the vehicle control compared with the corresponding value
din-1-yl)-3-phenylpropanamide (60). Carboxylic acid 59 at that time point for specimens containing the test com-
(886 mg, 3.42 mmol) was added to a 500 mL oven-dried flask pound. Assays were performed with n = 3, and concentration-
with stir bar and sealed under N2. Anhydrous DCM (150 mL), response curves were plotted using GraphPad Prism, with
HATU (1.95 g, 5.13 mmol), DIPEA (655 μL, 3.76 mmol), and N
N-diethyethylenediamine (624 μL, 4.44 mmol) were sequen-
tially added and the solution was stirred at 20 °C for 12 h.
̲
error bars representing standard error of the mean (SEM).
Crystallographic data
After 12 h, the reaction was concentrated under vacuum, dry CCDC 2004020 and 2004021 (Scheme 9), 2004022 (Scheme 12),
loaded using celite, and purified via flash chromatography and 2004023 (Fig. 3) contain the supplementary crystallo-
(30 g C18, 0–95% MeOH/H2O w/0.1% formic acid) to afford graphic data for this paper.†
amide 60 as an off-white waxy solid (918 mg, 75%).
1
m.p. 75–77 °C; H NMR (400 MHz, CD3OD) δ 7.44 (dd, J = 6.9,
1.5 Hz, 1H), 7.34 (ddd, J = 9.0, 6.7, 2.0 Hz, 1H), 7.28–7.26 (m, Author contributions
2H), 7.24–7.19 (m, 3H), 6.36 (dd, J = 9.0, 0.6 Hz, 1H), 6.14 (td, J
= 6.7, 1.3 Hz, 1H), 5.39 (d, J = 9.5 Hz, 1H), 5.19 (d, J = 9.5 Hz,
Proposed synthetic routes: C. D., E. G. Synthesized and charac-
terized compounds: E. G. Conducted the insulin reduction
assay: C. S., L. L., R. F. Determined X-ray crystal structures:
S. V. L. Wrote and edited the manuscript: E. G., C. D.
1H), 3.79–3.73 (m, 1H), 3.64–3.57 (m, 1H), 3.38–3.22 (m, 6H),
1.31 (t, J = 7.3 Hz, 6H); 13C NMR (100 MHz, CD3OD) δ 171.8,
164.4, 142.5, 141.1, 140.0, 129.5, 129.4, 128.0, 120.5, 108.4,
73.4, 67.3, 53.0, 49.1, 35.7, 9.2. LC-MS tR = 1.07, 1.32; m/z =
357.95 (M + H).
N-[2-(Diethylamino)ethyl]-3-oxo-2-(2-oxo-1,2-dihydropyridin-1-
Funding sources
yl)-3-phenylpropanamide (61). Alcohol 60 (47.5 mg,
C. D. and E. G. thank the Foundation for Women’s Wellness
0.133 mmol) was added to a 20 mL oven-dried vial with stir
for supporting this project in part. E. G. thanks Arthur
bar and sealed under N2. Anhydrous DCM (6.0 mL) and DMP
J. Schmitt and Marquette University for
a
graduate
(84.5 mg, 0.199 mmol) were sequentially added and the reac-
tion mixture was stirred at 20 °C for 1 h. After 1 h, H2O (2.7 μL,
0.15 mmol) was added via microsyringe. The reaction solution
was stirred at room temperature for 48 h before being
quenched with saturated Na2S2O3 (3 mL). The product was
extracted with DCM (3 × 10 mL) and concentrated. The crude
fellowship. R. F. thanks the National Heart, Lung, and Blood
Institute (R35HL135775) for support.
Notes
product was dissolved in DCM and purified via flash chrom- An initial version of this manuscript was posted to the preprint
atography (12 g SiO2, 0–20% MeOH/DCM) to afford ketone 61 server ChemRxiv.21
as an off-white solid (11.7 mg, 25%). Rf = 0.71 (80 : 20
1
DCM : MeOH); m.p. 124–129 °C; H NMR (400 MHz, CDCl3) δ
8.01 (d, J = 7.1 Hz, 2H), 7.65 (dd, J = 6.8, 1.7 Hz, 1H), 7.60–7.56 Conflicts of interest
(m, 1H), 7.48–7.44 (m, 1H), 7.40–7.34 (m, 2H), 6.60 (d, J = 9.2
There are no conflicts to declare.
Hz, 1H), 6.24 (td, J = 6.8, 1.3 Hz, 1H), 3.39–3.28 (m, 2H),
2.57–2.48 (m, 6H), 0.96 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 192.0, 165.2, 162.0, 140.7, 137.4, 135.0, 134.3, 129.1,
Acknowledgements
128.8, 119.9, 106.2, 61.6, 51.1, 46.7, 37.4, 11.5; LC-MS tR = 1.57;
m/z = 356.20 (M + H).
We thank Dr Sheng Cai for assistance with LC-MS, GC-MS, and
NMR instruments, Dr. Chris Kelly for a sample of Bobbitt’s
PDI assay protocol
salt, and ChemAxon Ltd for access to chemical property and
The PDI-catalyzed reduction of insulin was assayed by measur-
structure prediction software.
ing the increase in turbidity as detected at an optical density
of 650 nm using a Spectramax M3 (Molecular Devices,
Sunnyvale, CA). Compounds were assayed in a clear, flat-
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insulin, 2 mM EDTA and 0.5 mM DTT (all purchased from
Sigma Aldrich, St Louis, MO). Compounds were used at the
concentrations indicated. The reaction was performed at 25 °C
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