Improved Synthesis of a Cyclic Glutamine Analogue Used in Antiviral Agents Targeting 3C and 3CL Proteases including SARS-CoV-2 Mpro

Article abstract of DOI:10.1021/acs.joc.1c01299

An intermediate in the synthesis of numerous antiviral protease inhibitors is the glutamine analogue, (3S)-pyrrolid-2-one-3-yl-l-alanine. Preparations of compounds based on this pharmacophore are hindered by the lack of a reliably high yielding synthesis

Full text of DOI:10.1021/acs.joc.1c01299

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Note
Improved Synthesis of a Cyclic Glutamine Analogue Used in
Antiviral Agents Targeting 3C and 3CL Proteases Including SARS-
CoV‑2 Mpro
Wayne Vuong and John C. Vederas*
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ABSTRACT: An intermediate in the synthesis of numerous
antiviral protease inhibitors is the glutamine analogue, (3S)-
pyrrolid-2-one-3-yl-L-alanine. Preparations of compounds based on
this pharmacophore are hindered by the lack of a reliably high
yielding synthesis of protected forms of this amino acid. We
describe an improved scalable route with readily available reagents
and facile purification. This methodology employs γ-allylation of
dimethyl N-BocGlu, further Boc N-protection, OsO4-periodate oxidation, O-Me oxime formation, and RaNi-catalyzed
hydrogenolysis with concomitant cyclization under basic conditions.
any positive strand RNA viruses of the Picornaviridae
and Coronaviridae families require a cysteine protease,
interaction of the side chain amide moiety with electrophilic
groups intended to interact with the cysteine thiol of the target
protein. Reported yields for the synthesis of this building block
have varied, typically from 27% to 66% overall
yield19,21−23,20,24 starting from N-Boc-L-glutamate dimethyl
ester (Scheme 1). The original kilogram-scale synthesis
M
typically designated as 3C or 3CL, to specifically process an
initially synthesized protein through cleavage at glutamine
(Gln) residues.1−5 This process produces the protein frag-
ments essential for viral replication. Well known picornaviruses
include poliovirus, human rhinovirus, and foot and mouth
disease virus.1 The coronavirus group has many animal and
human pathogens, notably the severe acute respiratory
syndrome 2 (SARS-CoV-2) virus that causes COVID19.4−8
Inhibition of its main chymotrypsin-like cysteine (3CL)
protease, known as Mpro, is a key objective for development
of drugs to combat this disease.2,6,8−18 A key building block for
many such protease inhibitors (Figure 1) is the cyclic
glutamine analogue, (3S)-pyrrolid-2-one-3-yl-L-alanine
(1).2,6,8,9,11,12,14,17−20
Scheme 1. Standard Synthesis of Glutamine Analogue 1
developed by Pfizer Global Research and Development
reported an overall yield of 40% over 3 steps19a testament
to the difficulties associated with this seemingly facile process.
Despite some reports of higher yields,24 in our hands, ∼40%
is generally a maximum. The protected (3S)-pyrrolid-2-one-3-
yl-L-alanine is commercially available from some fine chemical
suppliers, typically at a cost of ca. $150 for 50 mg. In the
present work, we report a slightly longer (5 steps), but higher
yielding, procedure to generate this amino acid in suitably
protected form that requires minimal purification and appears
easily scalable to make larger quantities.
Its constrained structure binds well to viral protease residues
required for recognition of the cleavage site. It also prevents
Received: June 3, 2021
Figure 1. Select viral inhibitors containing a cyclic glutamine
analogue, incorporated via utilization of compound 1. Substructures
in inhibitors derived from compound 1 are denoted in red.
© XXXX The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.joc.1c01299
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The previously reported synthetic pathway in Scheme 1
begins with the stereoselective cyanomethylation of the γ-
carbon of Boc-L-glutamate dimethyl ester. This method was
initially developed by Hanessian and co-workers25,26 and later
modified by Tian et al.19 This is followed by a reduction of the
cyano group to a primary amine via hydrogenation in the
presence of PtO2 or borohydride reduction utilizing NaBH4 in
the presence of CoCl2. Subsequent cyclization onto the
adjacent methyl ester to form the five-membered lactam ring
then furnishes the desired compound. A major point of
product loss is the initial alkylation step. The Hanessian group
had reported that allylation provided much higher yields,
especially with allyl bromide,25 compared to alkylation with
bromoacetonitrile. Hence, we developed the route shown in
Scheme 2 to make the bis-N-Boc protected (3S)-pyrrolid-2-
one-3-yl-L-alanine methyl ester (6).
showed that hydrogenolysis at higher pressures using more
dilute substrate concentrations afforded cleaner transforma-
tion. We hypothesize that this is due to a more rapid reduction
combined with diminished intermolecular interactions that
could result in dimerization events. Furthermore, RaNi has a
distinct advantage in the large scale synthesis because the
catalyst can be cleanly removed by employment of a magnetic
source.28 Concurrent with the reduction step, cyclization can
be achieved in the presence of NaHCO3 and 15-crown-5 ether,
cleanly producing cyclized product 6 in quantitative yield.
We next examined this synthetic sequence on multi-gram
scale. Allylation of Boc-glutamic acid dimethyl ester (10 g)
gave alkene 2 in somewhat lower 90% yield after purification
by flash chromatography. The di-Boc protection proceeded in
nearly quantitative yield to furnish 3, which was efficiently
purified via a small silica gel plug. Lemieux−Johnson oxidation
of this compound then furnished aldehyde 4 in 96% isolated
yield after filtration through a silica plug. Oxime ether 5 was
obtained as pure material by extraction and aqueous washing
in quantitative yield. Hydrogenolysis of 5 with RaNi gave
glutamine analogue 6 in 91% yield after a rapid purification by
flash chromatography. To confirm the utility of 6 as an
alternative to the mono-Boc derivative 1, it was deprotected
with TFA and directly coupled to N-Cbz-Leu-OH to form the
corresponding known7−9 dipeptide 7 in 89% isolated yield. As
expected, the process proceeds in comparable yield.
Scheme 2. Synthesis of Glutamine Analogue 6
In summary, an improved synthetic route is described to a
protected derivative 6 of (3S)-pyrrolid-2-one-3-yl-L-alanine. In
comparison to the benchmark 40% overall yield to 1 starting
with Boc-glutamic acid dimethyl ester, the synthetic route
presented here reliably furnishes 6 in 79−86% overall yield
with minimal purification. It employs inexpensive, readily
available reagents with the use of only catalytic quantities of
more valuable transition metals (e.g., OsO4). With the
widespread utilization of this glutamine pharmacophore in
numerous antiviral compounds, this route can facilitate
production of drug candidates that bind to viral cysteine
proteases and expedite efforts toward the design of new
antivirals.
Smaller scale (750 mg) synthesis utilizing allyl bromide as
previously described25 gives alkene 2 in 96% isolated yield.
Direct oxidative cleavages of the alkene to an aldehyde are low
yielding and generate an array of undesired products, as is
typical for glutamate and aspartate aldehyde derivatives when
there is a single protecting group on the amide nitrogen.27
Hence, the nitrogen atom was further Boc protected in 92%
yield to provide 3. Although ozonolysis of 3 generates side
products, Lemieux−Johnson oxidation with catalytic quantities
of OsO4 (2 mol %) in the presence of NaIO4 and 2,6-lutidine
afforded aldehyde 4 in quantitative yield.
EXPERIMENTAL SECTION
Product Characterization. Nuclear magnetic resonance (NMR)
spectra were obtained using an Agilent VNMRS 700 MHz or Agilent/
Varian VNMRS 500 MHz spectrometers. For H spectra, δ values
■
1
1
At this point, the aldehyde had to be transformed into a
primary amine for ring closure. Several approaches were
attempted, including direct reductive amination using
ammonia, imine formation using benzyl amine, followed by
catalytic reduction, and oxime/oxime ether formation,
followed by N−O bond cleavage and reduction. The latter
proved to be best. Initial use of hydroxylamine hydrochloride
formed the desired oxime in modest yields (∼70%) with side
products, rendering it less suitable for scale-up. As an
alternative, O-benzylhydroxylamine and O-methylhydroxyl-
amine were examined. O-Benzylhydroxylamine formed the
desired oxime ether in 89% isolated yield, whereas reaction
with O-methylhydroxylamine gave 5 in quantitative yield.
Reduction of the methyl oxime ether 5 was attempted using a
number of methods. Reductive cleavage via hydrogenation
using Pd/C is a common approach, but generally requires
acidic conditions, which are incompatible with the Boc
protection. However, Raney Nickel (RaNi) successfully
reduced the methyl oxime to a primary amine. Optimization
were referenced to CDCl3 (7.26 ppm), and for 3C spectra, δ values
were referenced to CDCl3 (77.16 ppm) the solvent. Signal
assignments were confirmed using 2D NMR methods including
COSY, HSQC, and HMBC as required. Infrared spectra (IR) were
recorded on a Nicolet Magna 750 or a 20SX FT-IR spectrometer.
Cast film and thin film refer to the evaporation of a solution on a
NaCl plate. Mass spectra were recorded on a ZabSpec IsoMass VG
(high-resolution electrospray (ES)). LC-MS analysis was performed
on an Agilent Technologies 6220 orthogonal acceleration TOF
instrument equipped with +ve and −ve ion ESI ionization, and full-
scan MS (high-resolution analysis) with two-point lock mass
correction operating mode. The instrument inlet was an Agilent
Technologies 1200 SL HPLC system.
Reagents and Solvents. All commercially available reagents and
protected amino acids were purchased and used without further
purification unless otherwise noted. All the solvents used for reactions
were used without further purification unless otherwise noted. Dry
solvents refer to solvents freshly distilled over appropriate drying
reagents prior to use. Commercially available ACS grade solvents
(>99.0% purity) were used for column chromatography without any
further purification.
B
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Purification. All reactions and fractions from column chromatog-
raphy were monitored by thin layer chromatography (TLC) using
glass plates (5 × 2.5 cm) precoated (0.25 mm) with silica gel (normal
SiO2, Merck 60 F254). Visualization of TLC plates was performed by
UV fluorescence at 254 nm in addition to staining by KMnO4. Flash
chromatography was performed using Merck type 60, 230−400 mesh
silica gel at elevated pressures.
HRMS (ESI) Calcd for C15H25NNaO6 [M + Na]+ 338.1574, found
338.1570.
Dimethyl (2S,4S)-2-Allyl-4-(bis(tert-butoxycarbonyl)amino)-
pentanedioate (3).
Synthesis. Dimethyl (2S,4S)-2-Allyl-4-((tert-butoxycarbonyl)-
amino)pentanedioate (2).
Small Scale. This procedure was adapted from the literature.27,29
Compound 2 (3.00 g, 9.51 mmol, 1.0 equiv) was dissolved in 31.7 mL
of MeCN in a flame-dried 100 mL round-bottom flask. To this were
successively added DMAP (0.230 g, 1.90 mmol, 0.2 equiv) and Boc2O
(8.30 g, 38.1 mmol, 4.0 equiv). The reaction mixture then quickly
changed color to a light orange and was allowed to stir at rt. A check
by TLC at 94 h showed near total consumption of starting material.
The reaction mixture was then concentrated in vacuo to ca. 10 mL to
remove the MeCN. After concentration to a minimal volume, the
material was partitioned between ca. 75 mL each of EtOAc and 1 M
citric acid, then separated. The EtOAc layer was washed once more
with H2O, then once with brine. The EtOAc layer was then dried over
Na2SO4 and concentrated to furnish a crude, dark orange oil. This
material was purified by flash chromatography using an eluent system
of 15/85 EtOAc/Hexane. Product elution was monitored by TLC
and KMnO4 staining (Rf = 0.29, 15/85 EtOAc/Hexane). Concen-
tration of product fractions furnishes a yellow oil as the desired
product (3.63 g, 8.74 mmol, 92% yield).
Large Scale. Compound 2 (9.500 g, 30.12 mmol, 1.0 equiv) was
deposited in a flame-dried 250 mL round-bottom flask and dissolved
in 100 mL of freshly distilled MeCN. DMAP (0.740 g, 6.02 mmol, 0.2
equiv) and Boc2O (26.30 g, 120.5 mmol, 4.0 equiv) were then
sequentially added under an argon atmosphere. The reaction mixture
was then capped under an argon atmosphere and allowed to stir at rt,
changing to a yellow color. After 99 h, a TLC check of the reaction
mixture showed total consumption of starting material. The MeCN
was removed by concentrating the reaction mixture in vacuo to a
minimal volume (ca. 30 mL) and was then partitioned between ca.
250 mL each of EtOAc and 1 M citric acid. The layers were separated,
and the aqueous layer was then extracted with additional EtOAc (2×).
The combined EtOAc layers were then washed with H2O (1×) and
brine (1×), then dried over Na2SO4. Concentration in vacuo then
furnishes a dark yellow oil. A TLC of this crude material showed only
product (Rf = 0.29, 15/85 EtOAc/Hexane, KMnO4 staining), along
with a spot on the baseline. The crude oil (crude mass ca. 15 g) was
deposited onto a silica plug (silica mass ca. 135 g) and eluted with
1.25 L of eluent (15/85 EtOAc/Hexane). Total elution was
monitored by TLC. Concentration of product fractions furnished a
yellow oil as the desired product (12.51 g, 30.12 mmol, >99% yield).
IR (DCM cast film, νmax/cm−1) 3079, 2981, 2953, 1715, 1717,
1703, 1369, 1167, 1145.
Small Scale. This procedure was adapted from the literature.25
Commercially available Boc-L-glutamate dimethyl ester (0.750 g, 2.74
mmol, 1.0 equiv) was dissolved in 11.0 mL of freshly distilled THF in
a flame-dried 50 mL round-bottom flask under an argon atmosphere.
The solution was then cooled to −78 °C, and LiHMDS (1 M in THF,
5.92 mL, 5.92 mmol, 2.16 equiv) was added dropwise over 5 min. The
reaction mixture was then allowed to incubate at −78 °C for an
additional 1 h. Allyl bromide (0.710 mL, 8.22 mmol, 3.0 equiv) was
then added dropwise over a period of 30 min, and the reaction
mixture became a light yellow color. The reaction mixture was
checked by TLC after 2 h, and the starting material was found to be
fully consumed. The reaction was quenched via addition of HCl (1 M,
7.5 mL) and subsequently extracted with EtOAc (3×). The combined
EtOAc layers were washed with H2O (2×) and brine (1×). The
organic layer was then dried over MgSO4, filtered, and concentrated
to furnish a light yellow oil as a crude. The material was then purified
by flash chromatography over silica, using an eluent system of 15/85
EtOAc/Hexane. Product elution was monitored by KMnO4 staining
(Rf = 0.44, 30/70 EtOAc/Hexane). Concentration of product
fractions furnishes a clear, slightly yellow oil as the desired compound
(0.830 g, 2.63 mmol, 96% yield).
Large Scale. Commercially available Boc-L-glutamate dimethyl
ester (10.00 g, 36.32 mmol, 1.0 equiv) was dissolved in 134.5 mL of
freshly distilled THF under an argon atmosphere in a flame-dried 500
mL round-bottom flask. The reaction mixture was then cooled to −78
°C, and LiHMDS (1 M in THF, 79.90 mL, 79.90 mmol, 2.2 equiv)
was added dropwise. The reaction mixture was then allowed to stir at
−78 °C for 1 h. Next, allyl bromide (9.42 mL, 109 mmol, 3.0 equiv)
was added dropwise over a period of 15 min. The reaction mixture
was then stirred at −78 °C for 4 h, and a check by TLC showed
minimal starting material remaining at the end of this period. The
reaction mixture was then diluted with 75 mL of sat. NH4Cl(aq) to
quench the reaction. The reaction mixture was then warmed to rt and
diluted with 75 mL of EtOAc. The layers were separated, and the
aqueous layer was then extracted with additional EtOAc (2×). The
combined EtOAc layers were washed with H2O (2×) and brine (1×),
then dried over MgSO4. Filtration and subsequent concentration of
the organic layer then furnish a yellow oil as a crude product. The
material was purified by flash chromatography over silica using an
eluent system of 15/85 EtOAc/Hexane. Product elution was
monitored by KMnO4 (Rf = 0.15, 15/85 EtOAc/Hexane).
Concentration of product fractions furnishes a light yellow oil as
the desired product (10.34 g, 32.78 mmol, 90% yield).
1H NMR (700 MHz, CDCl3) δH 5.73 (1H, ddt, J = 16.9, 9.9, 7.0
Hz, H9), 5.07 (1H, dq, J = 17.2, 1.6 Hz, H10), 5.05−5.01 (1H, m,
H10), 4.96 (1H, dd, J = 8.9, 5.5 Hz, H5), 3.71 (3H, s, H7), 3.65 (3H,
s, H1), 2.59 (1H, ttt, J = 14.1, 8.26, 5.9 Hz, H3), 2.39−2.33 (1H, m,
H4), 2.31−2.23 (3H, m, H4, H8), 1.50 (18H, s, 6× Boc-CH3).
13C NMR{1H} (175 MHz, CDCl3) δc 175.4 (C2), 171.1 (C6),
151.8 (Boc-CO), 134.9 (C9), 117.4 (C10), 83.3 (Boc-4 °C), 56.6
(C5), 52.3 (C7), 51.7 (C1), 42.8 (C3), 36.7 (C4), 32.2 (C8), 28.0
(Boc-CH3).
IR (DCM cast film, νmax/cm−1) 3370, 3080, 2979, 2954, 1739,
1718, 1516, 1440, 1169.
1H NMR (700 MHz, CDCl3) δH 5.75−5.65 (1H, m, H9), 5.11−
5.02 (2H, m, H10), 4.95 (1H, d, NH), 4.41−4.29 (1H, m, H5), 3.72
(3H, s, H7), 3.66 (3H, s, H1), 2.57 (1H, app quint, H3), 2.41−2.27
(2H, m, H4), 2.00 (2H, app t, H8), 1.44 (9H, s, 3× Boc-CH3).
13C NMR{1H} (175 MHz, CDCl3) δc 175.5 (C2), 172.9 (C6),
155.4 (Boc-CO), 134.4 (C9), 117.7 (C10), 80.1 (Boc-4 °C), 52.4
(C7), 52.2 (C5), 51.8 (C1), 41.9 (C3), 36.5 (C4), 33.8 (C8), 28.3
(Boc-CH3).
26
OR [α]D = −21.81 (c = 0.54, DCM).
HRMS (ESI) Calcd for C20H33NNaO8 [M + Na]+ 438.2098, found
26
OR [α]D = +74.42 (c = 0.23, DCM).
438.2091.
C
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Dimethyl (2S,4R)-2-(Bis(tert-butoxycarbonyl)amino)-4-(2-
Small Scale. Compound 4 (0.050 g, 0.120 mmol, 1.0 equiv), MeO-
NH2·HCl (0.012 g, 0.144 mmol, 1.2 equiv), and NaOAc (0.020 g,
0.240 mmol, 2.0 equiv) were charged into a 5 mL round-bottom flask,
to which dry DCM (0.3 mL) was added. The flask was capped and
stirred at rt for 16 h. A check by TLC (25/75 EtOAc/Hexane) at this
point showed total consumption of starting material. The reaction
mixture was then quenched by addition of ca. 5 mL of sat. NaHCO3,
followed by dilution with ca. 5 mL of EtOAc. The layers were then
separated, and the aqueous layer was further extracted with EtOAc
(2×). The combined EtOAc layers were washed sequentially with sat.
NaHCO3 (2×), H2O (2×), and brine (1×), then dried over Na2SO4.
Concentration of the EtOAc solution then furnishes a yellow oil as a
crude. The material was found to not require further purification (Rf =
0.28, 25/75 EtOAc/Hexane). Concentration of product fractions
furnishes a clear, colorless oil as the desired product (mixture of imine
isomers, 0.054 g, 0.120 mmol, >99% yield).
oxoethyl)pentanedioate (4).
Small Scale. This procedure was adapted from the literature.30
Compound 3 (0.166 g, 0.400 mmol, 1.0 equiv) was dissolved in a
mixture of dioxane/H2O (3:1, 4.00 mL) in a 25 mL round-bottom
flask. To this solution were added 2,6-lutidine (0.093 mL, 0.800
mmol, 2.0 equiv), OsO4 (0.002 g, 0.008 mmol, 0.02 equiv), and
NaIO4 (0.342 g, 1.60 mmol, 4.0 equiv) in succession. The reaction
mixture was then capped with a septa and allowed to stir at rt, quickly
turning into a slurry. After 2 h, the reaction mixture was checked by
TLC 30/70 EtOAc/Hexane), showing consumption of all starting
material. The reaction mixture was then diluted with ca. 25 mL each
of DCM and H2O, then separated. The H2O layer was further
extracted with DCM (2×), and the combined DCM layers were
washed with brine (1×). The DCM layer was dried over Na2SO4 and
concentrated to furnish a crude that turned black in coloration. The
crude material was purified via flash chromatography using an eluent
system of 20/80 EtOAc/Hexane. The black impurity was found to
stick to the baseline of the silica. Product elution was monitored by
TLC and KMnO4 staining (Rf = 0.29, 30/70 EtOAc/Hexane).
Concentration of product fractions and co-evaporation with Et2O
furnishes an oil (0.167 g, 0.400 mmol, >99% yield).
Large Scale. Compound 4 (4.18 g, 10.00 mmol, 1.0 equiv), MeO-
NH2·HCl (1.00 g, 12.00 mmol, 1.2 equiv), and NaOAc (1.64 g, 20.00
mmol, 2.0 equiv) were added to a 50 mL round-bottom flask and
dissolved with freshly distilled DCM (11.7 mL). The vessel was then
capped with a septa and stirred at rt for 16 h. A check by TLC (25/75
EtOAc/Hexane) showed total consumption of starting material, so
the reaction mixture was diluted with ca. 150 mL each of EtOAc and
sat. NaHCO3, then separated. The aqueous layer was further extracted
with EtOAc (2×). The combined EtOAc layers were then washed
with sat. NaHCO3 (2×), H2O (2×), and brine (1×). The EtOAc
layer was then dried over Na2SO4 and concentrated to furnish a
yellow oil (4.47 g, 10.00 mmol, >99% yield). A check of this crude
material by NMR showed that it is quite clean and suitable for
subsequent use without further purification.
Large Scale. Compound 3 (12.00 g, 28.88 mmol, 1.0 equiv) was
dissolved in a mixture of dioxane/H2O (3:1, 290 mL) in a 500 mL
round-bottom flask. To this solution were added 2,6-lutidine (6.69
mL, 57.8 mmol, 2.0 equiv), OsO4 (0.147 g, 0.578 mmol, 0.02 equiv),
and NaIO4 (24.71 g, 115.5 mmol, 4.0 equiv) in succession, and the
reaction mixture was capped with a septa. A check by TLC (30/70
EtOAc/Hexane) after 3 h showed complete consumption of starting
material. The reaction mixture was then diluted with ca. 1 L each of
DCM and H2O, and the layers were separated. The H2O layer was
further extracted with DCM (2×), and the combined DCM layers
were washed with brine. Drying over Na2SO4 and concentration
furnished a crude dark brown oil. The crude material (crude mass ca.
25 g) was loaded onto a silica plug (silica mass ca. 130 g) and eluted
using 1.4 L of 15/85 EtOAc/Hexane, with elution completion being
confirmed by TLC (Rf = 0.29, 30/70 EtOAc/Hexane). Concentration
of product solution furnishes a brown-black oil after co-evaporation
with Et2O (11.57 g, 27.71 mmol, 96% yield).
IR (DCM cast film, νmax/cm−1) 2981, 2953, 1797, 1744, 1437,
1369, 1274, 1169, 1146.
1H NMR (700 MHz, CDCl3) δH 7.33 (0.6H, app t, J = 6.0 Hz, H9,
Isomer A), 6.67 (0.4H, app t, J = 5.4 Hz, H9, Isomer B), 5.02−4.97
(1H, m, H5), 3.86 (1.3H, s, H10, Isomer B), 3.78 (1.7H, s, H10,
Isomer A), 3.71 (3H, s, H7), 3.68 (1.3H, s, H1, Isomer B), 3.67
(1.7H, s, H1, Isomer A), 2.78−2.69 (1H, m, H3), 2.68−2.39 (2H, m,
H8?), 2.36−2.23 (2H, m, H4), 1.50 (18H, s, 6× Boc-CH3).
13C NMR{1H} (175 MHz, CDCl3) δc 174.7 (C2, Isomer B), 174.7
(C2, Isomer A), 170.9 (C6, Isomer B), 170.9 (C6, Isomer A), 151.7
(Boc-CO), 148.0 (C9, Isomer B), 147.4 (C9, Isomer A), 83.6
(Boc-4 °C), 61.8 (C10, Isomer B), 61.5 (C10, Isomer A), 56.4 (C5,
Isomer B), 56.3 (C5 Isomer A), 52.3 (C7), 52.1 (C1, Isomer B), 52.0
(C1, Isomer A), 40.6 (C3, Isomer A), 40.2 (C3, Isomer B), 32.3 (C4,
Isomer B), 32.2 (C4, Isomer A), 31.9 (C8, Isomer A), 28.1 (C8,
Isomer B), 28.0 (Boc-CH3).
26
OR [α]D = −20.24 (c = 0.99, DCM).
IR (DCM cast film, νmax/cm−1) 2981, 2945, 1795, 1743, 1703,
1437, 1369, 1251, 1170, 1146, 1122.
HRMS (ESI) Calcd for C20H34N2NaO9 [M + Na]+ 469.2157,
found 469.2153.
1H NMR (700 MHz, CDCl3) δH 9.74 (1H, s, H9), 4.99 (1H, dd, J
= 9.3, 5.5 Hz, H5), 3.71 (3H, s, H7), 3.69 (3H, s, H1), 2.99−2.94
(1H, m, H3), 2.88 (1H, ddd, J = 18.1, 9.1, 1.1 Hz, H8), 2.71 (1H, dd,
J = 18.1, 4.4 Hz, H8), 2.36−2.30 (1H, m, H4), 2.29−2.24 (1H, m,
H4), 1.49 (18H, s, 6× Boc-CH3).
Methyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrro-
lidin-3-yl)propanoate (6).
13C NMR{1H} (175 MHz, CDCl3) δc 199.0 (by HSQC, C9),
174.7 (C2), 170.8 (C6), 151.9 (Boc-CO), 83.6 (Boc-4 °C), 55.9
(C5), 52.4 (C7), 52.2 (C1), 45.1 (C8), 36.4 (C3), 32.0 (C4), 28.0
(Boc-CH3).
26
OR [α]D = −25.54 (c = 0.40, DCM).
Small Scale. This procedure was adapted from the literature.31,32
Compound 5 (0.080 g, 0.179 mmol, 1.0 equiv), Raney Nickel (W.R.
Grace and Co. Raney 2800, ca. 0.080 g), NaHCO3 (saturated solution
at 21 °C, 0.16 mL, ca. 1 equiv), 15-crown-5 ether (0.036 mL, 0.179
mmol, 1.0 equiv), and MeOH (7.0 mL) were added to a vial
(Chemglass CG4907-02) with a stir bar and custom cap equipped
with a glass frit. The mixture was then placed in a high pressure
reaction vessel (Parr Series 4750, 200 mL), stirred, and subjected to
purge-charge cycles (3×) with H2. The reaction mixture was then
subjected to H2 (800 psi, 41372 Torr) for 17.5 h. A check of the
reaction mixture at this point by LCMS (see Supporting Information)
HRMS (ESI) Calcd for C19H31NNaO9 [M + Na]+ 440.1891, found
440.1888.
Dimethyl (2S,4R)-2-(Bis(tert-butoxycarbonyl)amino)-4-(2-
(methoxyimino)ethyl)pentanedioate (5).
D
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Note
showed consumption of starting material. The catalyst was removed
using a magnetic source (computer hard-drive magnet), and the
reaction mixture was then partitioned between EtOAc and H2O, and
the layers separated. The H2O layer was further extracted with EtOAc
(2×), and the combined EtOAc layers were washed with brine (1×).
Drying over Na2SO4 and removal of solvent furnish a yellow crude.
This material was purified by flash chromatography, using an eluent
system of 85/15 EtOAc/Hexane. Product elution was monitored by
TLC and KMnO4 staining (Rf = 0.21, 85/15 EtOAc/Hexane, requires
prolonged heating to visualize with KMnO4). Concentration of
product fractions furnishes a clear, colorless oil (0.069 g, 0.179 mmol,
>99% yield).
Cbz-Leu-OH (90% pure, 0.023 g, 0.078 mmol, 1.0 equiv) and
HATU (0.030 g, 0.078 mmol, 1.0 equiv) were dissolved in 0.35 mL of
DMF. To this were added HOAT (0.6 M solution in DMF, 0.013 mL,
0.008 mmol, 0.1 equiv) and finally DIPEA (0.041 mL, 0.233 mmol,
3.0 equiv). The mixture turned yellow at this point, forming <Solution
2>, and was allowed to incubate at rt for 5 min.
<Solution 1> was then added to <Solution 2>, and the reaction
mixture was allowed to stir at rt for 1.5 h. The reaction mixture was
then diluted with 7.5 mL each of EtOAc and H2O. The layers were
separated, and the aqueous layer was further extracted with EtOAc
(2×). The combined EtOAc layers were washed sequentially with sat.
NaHCO3 (2×), 1 M HCl (2×), and brine (1×). Drying over Na2SO4,
followed by filtration and concentration, furnishes a crude yellow oil.
The material was then purified by flash chromatography using EtOAc
as the eluent. Product elution was monitored by TLC and KMnO4
staining (Rf = 0.47, 5/95 MeOH/EtOAc). Concentration of product
fractions furnishes a transparent oily film as the product (0.030 g,
0.069 mmol, 89%). All characterization data were found to match
previously reported values within experimental error.8
Large Scale. Compound 5 (6.00 g, 13.4 mmol, 1.0 equiv), Raney
Nickel (W.R. Grace and Co. Raney 2800, ca. 6.00 g), NaHCO3
(saturated solution at 21 °C, 12.0 mL, ca. 1 equiv), and MeOH (538
mL) were added to a glass bottle equipped with a fritted cap. This
mixture was then placed within a high pressure reaction vessel, stirred,
and subjected to purge-charge cycles with H2 (3×). The reaction
mixture was then subjected to high pressure H2 (800 psi, 41372
Torr). A check of the reaction mixture at 40 h by LCMS indicated the
presence of fully reduced, but uncyclized, material. More NaHCO3
(0.79 g, 9.41 mmol, 0.7 equiv) was added to the reaction, at which
point intramolecular cyclization was found to proceed. The
cyclization event was found to be sluggish at times, possibly as a
result of localized effects, but the cyclization rate can be increased by
addition of NaHCO3 (up to 2.5 equiv) or by heating to 60 °C with no
detrimental effects. The reaction was continually monitored by
LCMS, and upon completion, the Raney Nickel was removed using a
magnetic source (computer hard-drive magnet) and the solution was
diluted with 1.5 L each of EtOAc and H2O. The layers were
separated, and the aqueous layer was further extracted using EtOAc
(2×). The combined organic layers were then washed with brine (1×)
and dried over Na2SO4. Concentration of the organic extracts then
furnishes a yellow oil as a crude. The oil was then subjected to flash
chromatography, using an eluent system of 85/15 EtOAc/Hexane.
Product elution was monitored by TLC and KMnO4 staining (Rf =
0.21, 85/15 EtOAc/Hexane, prolonged heating required for visual-
ization). Concentration of product fractions furnishes an oil (4.72 g,
12.20 mmol, 91% yield).
IR (DCM cast film, νmax/cm−1) 3286, 2956, 2926, 2689, 1537,
1439, 1268.
1H NMR (700 MHz, CDCl3) δH 7.88 (1H, d, J = 6.6 Hz, NH),
7.37−7.27 (5H, m, H1, H2, H3), 6.31 (1H, s, NH), 5.44 (1H, d, J =
8.3 Hz, NH), 5.09 (2H, s, H5), 4.50−4.42 (1H, m, H9), 4.39−4.31
(1H, m, H7), 3.72 (3H, s, H11), 3.35−3.23 (2H, m, H15), 2.47−2.30
(2H, m, H13, H14), 2.23−2.14 (1H, m, H12), 1.93−1.85 (1H, m,
H12), 1.85−1.77 (1H, m, H14), 1.77−1.70 (1H, m, H18), 1.70−1.63
(1H, m, H17), 1.56−1.47 (1H, m, H17), 1.0−0.91 (6H, m, H19).
13C{1H} NMR (175 MHz, CDCl3) δc 179.8 (C16), 171.9 (C8),
172.1 (C10), 152.1 (C6), 136.4 (C4), 128.5 (C2), 128.1 (C3), 128.0
(C1), 66.9 (C5), 53.4 (C7), 52.4 (C11), 51.6 (C9), 42.4 (C17), 40.5
(C15), 38.4 (C13), 32.9 (C12), 28.4 (C14), 24.7 (C18), 22.9 (H19),
22.1 (H19).
26
OR [α]D = −9.89 (c = 0.38, DCM).
HRMS (ESI) Calcd for C22H31N3NaO6 [M + Na]+ 456.2105,
found 456.2102.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.1c01299.
■
sı
IR (DCM cast film, νmax/cm−1) 3227, 2980, 2935, 1791, 1749,
1703, 1369, 1274, 1148, 1124.
1H NMR (700 MHz, CDCl3) δH 5.54 (1H, s, NH), 5.04 (1H, dd, J
= 10.2, 4.8 Hz, H4), 3.72 (3H, s, H6), 3.38−3.26 (2H, m,
diastereotopic H8), 2.50 (1H, ddd, J = 14.7, 10.1, 3.7 Hz,
diastereotopic H3), 2.41−2.32 (2H, m, H2, diastereotopic H7),
2.10 (1H, ddd, J = 14.8, 11.0, 4.8 Hz, diastereotopic H3), 1.88−1.78
(1H, m, diastereotopic H7), 1.51 (18H, s, 6× Boc-CH3).
Detailed experimental procedures and spectra of
products (PDF)
AUTHOR INFORMATION
Corresponding Author
■
13C NMR{1H} (175 MHz, CDCl3) δc 179.2 (C1), 171.2 (C5),
152.1 (Boc-CO), 83.5 (Boc-4 °C), 56.2 (C4), 52.3 (C6), 40.5
(C8), 37.9 (C2), 31.8 (C3), 28.1 (C7), 28.0 (Boc-CH3).
John C. Vederas − Department of Chemistry, University of
Alberta, Edmonton, Alberta, Canada T6G 2G2;
orcid.org/0000-0002-2996-0326; Email: john.vederas@
ualberta.ca
26
OR [α]D = −30.29 (c = 0.27, DCM).
HRMS (ESI) Calcd for C18H30N2NaO7 [M + Na]+ 409.1945,
found 409.1943.
Author
Methyl (S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-4-methylpen-
tanamido)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (7).
Wayne Vuong − Department of Chemistry, University of
Alberta, Edmonton, Alberta, Canada T6G 2G2;
orcid.org/0000-0002-3439-8788
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c01299
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Compound 6 (0.030 g, 0.078 mmol, 1.0 equiv) was dissolved in 2.0
mL of 50/50 DCM/TFA and allowed to deprotect for 1.5 h. The
mixture was then concentrated by rotovap and co-evaporated with
DCM (5×) to remove residual TFA. This oily material was then
dissolved in 0.35 mL of DCM to furnish <Solution 1>.
The authors thank Ryan McKay, Mark Miskolzie, Randy
Whittal, and Béla Reiz (University of Alberta) for assistance
with spectroscopic analyses. Financial support from Canadian
Institutes of Health Research (CIHR COVID19 May 172645)
E
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