Synthesis and evaluation of cyclopentane-based muraymycin analogs targeting MraY

Article abstract of DOI:10.1016/j.ejmech.2021.113272

Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for the development of new antibiotics. In this regard, phospho-MurNAc-pentapeptide translocase (MraY), an essential enzyme in the early stages of peptid

Full text of DOI:10.1016/j.ejmech.2021.113272

European Journal of Medicinal Chemistry 215 (2021) 113272
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and evaluation of cyclopentane-based muraymycin analogs
targeting MraY
,
,
,
, 1, 2
Seung-Hwa Kwak a 1, Won Young Lim a 1, Aili Hao b 1, Ellene H. Mashalidis b
,
Do-Yeon Kwon a, Pyeonghwa Jeong a, Mi Jung Kim a, Seok-Yong Lee b **, Jiyong Hong a
,
, *
a Department of Chemistry, Duke University, Durham, NC, 27708, United States
b Department of Biochemistry, Duke University Medical Center, Durham, NC, 27710, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for
the development of new antibiotics. In this regard, phospho-MurNAc-pentapeptide translocase (MraY),
an essential enzyme in the early stages of peptidoglycan biosynthesis, has emerged as a promising new
antibiotic target. We recently reported the crystal structures of MraY in complex with representative
members of naturally occurring nucleoside antibiotics, including muraymycin D2. However, these
nucleoside antibiotics are synthetically challenging targets, which limits the scope of medicinal chem-
istry efforts on this class of compounds. To gain access to active muraymycin analogs with reduced
structural complexity and improved synthetic tractability, we prepared and evaluated cyclopentane-
based muraymycin analogs for targeting MraY. For the installation of the 1,2-syn-amino alcohol group
of analogs, the diastereoselective isocyanoacetate aldol reaction was explored. The structureeactivity
relationship analysis of the synthesized analogs suggested that a lipophilic side chain is essential for
MraY inhibition. Importantly, the analog 20 (JH-MR-23) showed antibacterial efficacy against Staphylo-
coccus aureus. These findings provide insights into designing new muraymycin-based MraY inhibitors
with improved chemical tractability.
Received 30 September 2020
Received in revised form
21 January 2021
Accepted 2 February 2021
Available online 6 February 2021
Keywords:
Antibiotics
Peptidoglycan
MraY
Muraymycin
Cyclopentane
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
is responsible for maintaining cell shape by stabilizing the mem-
brane against osmotic pressure. Hence, biosynthesis of peptido-
The global burden of multidrug-resistant infections is a major
threat to global public health [1]. A decline in chemical and phar-
maceutical research to develop new antibiotics has resulted in few
truly new antibiotics in the pipeline. At the same time, widespread
antibiotic resistance is steadily eroding the effectiveness of existing
treatment options. Therefore, an urgent need exists for new anti-
biotics with novel modes of action.
Peptidoglycan is a cross-linked polymer of carbohydrates and
amino acids that comprises the cell wall of both Gram-negative and
Gram-positive bacteria [2]. It is essential for bacterial survival as it
glycan is a well-established target for antibiotic development. The
late stages of peptidoglycan biosynthesis (i.e., cross-linking) have
been extensively explored, which has resulted in the development
of the penicillin and vancomycin classes of antibiotics. However,
the early stages have been underexplored despite the fact that
there are many natural product inhibitors targeting these stages.
Therefore, these early steps offer excellent opportunities for new
antibiotic development.
Among the enzymes involved in the early stages of peptido-
glycan biosynthesis, phospho-MurNAc-pentapeptide translocase
(MraY) is a member of the polyprenyl-phosphate N-acetylhexos-
amine 1-phosphate-transferase (PNPT) superfamily. MraY is an
integral membrane protein that catalyzes the first membrane step
of bacterial cell wall biosynthesis, the transfer of the peptidoglycan
precursor phospho-MurNAc-pentapeptide to the lipid carrier
undecaprenyl phosphate (C55eP) [3]. This is an essential membrane
step of peptidoglycan biosynthesis, and as a result, the inhibition of
MraY leads to cell lysis. Therefore, MraY has long been considered a
* Corresponding author.
** Corresponding author.
E-mail addresses: seok-yong.lee@duke.edu (S.-Y. Lee), jiyong.hong@duke.edu
(J. Hong).
1
S-H.K., W.Y.L., A.H., and E.H.M. contributed equally to this work.
Current address: Pfizer Global Research & Development, Eastern Point Road,
2
Groton, CT, 06340, United States.
https://doi.org/10.1016/j.ejmech.2021.113272
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
promising target for the development of new antibiotics [4e6].
However, despite many years of effort, the development of antibi-
otics targeting MraY has been stagnant largely due to insufficient
understanding of MraY structure, function, and inhibition. Recently,
we reported the crystal structures of Aquifex aeolicus MraY
(MraYAA) alone and in complex with representative members of the
liposidomycin/caprazamycin, capuramycin, and mureidomycin
classes of nucleoside inhibitors [7e9]. The analysis of these crystal
structures enhanced our understanding of the mechanisms of MraY
catalysis and inhibition by natural products and paved the way to
the development of new MraY-targeting antibiotics.
an aminopropyl linker to a urea peptide moiety consisting of
L-
leucine or -hydroxyleucine, -epicapreomycidine, and -valine. The
L
L
L
promising MraY inhibitory activity of muraymycins makes them
attractive candidates for future antibacterial agent development.
Since the first total synthesis of muraymycin D2 reported by
Ichikawa, Matsuda, and co-workers [11], there have been a number
of reports on the synthesis and biological evaluation of mur-
aymycins and muraymycin analogs [12e27]. However, despite
great efforts in the synthesis of muraymycins, they are still chal-
lenging synthetic targets. Moreover, the structureeactivity rela-
tionship (SAR) of muraymycins reported to date has primarily
focused on the peptide motif and the 50-position with the amino
ribose [16e18]; little is known about the role of the ribose core of
muraymycins in MraY inhibition [28]. Therefore, development of
muraymycin analogs with modifications on the ribose moiety
would help to elucidate the role of the ribose unit of muraymycins
in MraY inhibition.
Among the naturally occurring nucleoside antibiotics, the
muraymycins (Fig. 1a) were isolated from a broth of a Streptomyces
sp. and represent a promising class of new nucleoside antibiotics
targeting MraY [10]. They have a glycyl-uridine motif connected via
Towards this goal, we embarked on the synthesis of cyclo-
pentane core analogs of muraymycins. As the ribose of mur-
aymycins has no specific interaction with the amino acid residues
within the active site of MraY (Fig. 1b) [8], we reasoned that a
cyclopentane with a similar ring conformation as the tetrahydro-
furan ring of the ribose would be an excellent substitute for the
ribose moiety of muryamycins. More importantly, it is more
amenable to modifications with various substituents than the
ribose ring of muraymycins. Indeed, it has been reported that the
replacement of a ribose with a cyclopentane ring improved the
biological activity of the cyclopentane analog over the original
compound [29]. By substituting the ribose of muraymycins with a
cyclopentane ring, we anticipated to gain access to active mur-
aymycin analogs with reduced structural complexity and/or
improved synthetic tractability. Here we report the synthesis and
evaluation of cyclopentane core-based muraymycin analogs.
2. Results and discussion
2.1. Chemistry
The synthesis of the key intermediate 5 started with the
commercially available (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one
(2) (Scheme 1). Catalytic hydrogenation of 2 followed by N-Boc
protection gave the lactam 3 [30] (81% over two steps). Next, the
reductive ring cleavage of 3 with NaBH4 and boiling water catalyzed
neutral N-Boc deprotection [31] gave the known amine 4 [30] in
good yield (64% over two steps). To introduce a uracil group, we
coupled 4 with 3-ethoxyacryloyl isocyanate following the previ-
ously reported procedure [32]. The final acid-mediated cyclization
of the urea intermediate completed the synthesis of the key in-
termediate 5.
After the preparation of 5, the installation of the 1,2-syn-amino
alcohol moiety of muraymycins began with the oxidation of 5 to the
corresponding aldehyde. Initial attempts for the oxidation of 5 such
as Swern or PCC oxidation did not provide the desired aldehyde,
which led us to use of the IBX oxidation conditions reported by
Matsuda and co-workers [33]. The IBX oxidation of 5 smoothly
proceeded to provide the corresponding aldehyde, which was
converted to the
a,b-unsaturated ester 6 by treating with (tert-
butoxycarbonylmethylene)triphenylphosphorane. The Sharpless
aminohydroxylation reaction [33] of 6 was carried out with
(DHQD)2AQN as a chiral ligand to afford the 1,2-syn-amino alcohol
7
(46%) as a single diastereomer. Interestingly, the amino-
Fig. 1. (a) The structure of muraymycins. (b) Conformation of muraymycin D2 (1d,
green color) in the 1d-bound MraY structure (PDB: 5CKR). The hydrogen bond,
hydroxylation reaction of 6 did not give other diastereomeric 2,3-
amino alcohols as previously reported by others [34]. However,
the stereoselective aminohydroxylation reaction of 6 suffered from
the reproducibility and low-yield issues, which prompted us to
waterehydrogen bond,
pep stacking interaction, and salt bridge interaction between
1d and MraY are shown in green, cyan, magenta, and orange dashed lines, respectively.
Water molecules are presented as red spheres.
2

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
Scheme 1. Synthesis of cyclopentane-based MraY inhibitors 10 (JH-MR-21) and 11 (JH-
a
MR-22).a Reagents and conditions: (a) Pd/C, H2, MeOH, 25 ꢀC, 5 h; (b) Boc2O, DMAP,
MeCN, 25 ꢀC, 1 h, 81% for two steps; (c) NaBH4, MeOH, 25 ꢀC, 3 h, 80%; (d) H2O, reflux,
20 h, 80%; (e) 3-ethoxyacryloyl isocyanate, 4 Å MS, DMF, ꢁ20 to 25 ꢀC, 15 h, 49%; (f)
1 N H2SO4, reflux, 30 min, 68%; (g) IBX, MeCN, 80 ꢀC, 1.5 h; (h) Ph3P¼CHCO2t-Bu, THF,
0 ꢀC, 12 h, 45% for two steps; (i) CbzNH2, K2OsO2(OH)4, (DHQD)2AQN, t-BuOCl, n-PrOH,
0.6 N NaOH, 5 to 25 ꢀC, 2 h, 46% (dr > 20:1); (j) 8, NIS, TESOTf, CH2Cl2, 4 Å MS, 25 ꢀC,
30 min, 53%; (k) PPh3, THF/toluene (1/1), H2O, 25 ꢀC, 12 h, 63%; (l) 80% TFA in THF/H2O
(1/1), 25 ꢀC, 14 h; For 10: 41%; For 11: 91%.
explore other methods for the installation of the 1,2-syn-amino
alcohol moiety (vide infra). Next, the glycosylation reaction of 7
with various glycosyl donors and activators was explored. After an
extensive search for reaction conditions (see the Supplementary
Information for details), treatment of 7 with the n-pentenyl
glycoside 8 [35], TESOTf, and NIS provided the desired glycosylation
product in 53% as a single diastereomer. The azide group of the
glycosylation product was reduced under Staudinger’s conditions
to give the amine 9 (63%). The final global deprotection under acidic
conditions gave either the partially deprotected t-Bu ester 10 (JH-
MR-21) or the fully deprotected carboxylic acid 11 (JH-MR-22) as
the final product depending on purity of 9 and reaction time.
Since the lipophilic peptide chains of muraymycins play an
important role in MraY inhibition [11,16,18], we embarked on the
synthesis of a cyclopentane analog with a lipophilic side chain
(analog 20) starting from the common intermediate 5. As
mentioned above, when we prepared the cyclopentane-based an-
alogs 10 and 11, the Sharpless aminohydroxylation gave low yield
and inconsistent diastereoselectivity. To address these issues, we
turned our attention to the diastereoselective isocyanoacetate aldol
reaction reported by Dixon and co-workers [36] (Scheme 2). The
IBX oxidation of 5 followed by the coupling of the resulting alde-
hyde with methyl isocyanoacetate in the presence of chiral ami-
nophosphine ligand 12 and Ag2O proceeded to give a 2:1 mixture of
the aldol products 14a and 14b. The yield and stereoselectivity of
the isocyanoacetate aldol reaction were sensitive to catalyst acti-
vation time and temperature (see the Supplementary Information
Scheme 2. Synthesis of a cyclopentane-based MraY inhibitor with a lipophilic side
chain 20 (JH-MR-23).a
aReagents and conditions: (a) IBX, MeCN, 80 ꢀC, 1 h, 70%; (b) methyl isocyanoacetate,
Ag2O, 12, EtOAc, 4 Å MS, ꢁ78 to 0 ꢀC, 1.5 days; (c) 4 N HCl, THF, 25 ꢀC, 1 h; (d) CbzCl,
NaHCO3, THF/H2O (2/1), 0 ꢀC, 15 h, 20% for three steps; (e) 8, NIS, TESOTf, CH2Cl2,
4 Å MS, 25 ꢀC, 30 min, 60%; (f) Zn powder, NH4Cl, EtOH/H2O (3/1), 25 ꢀC, 1.5 h; (g)
Boc2O, NaHCO3, CH2Cl2, 25 ꢀC, 2 h, 52% for two steps; (h) Pd/C, H2, MeOH, 25 ꢀC, 3 h; (i)
16, NaBH3CN, HOAc, MeOH, 25 ꢀC, 15 h, 59% for two steps; (j) Zn powder, NH4Cl, MeOH,
25 ꢀC, 25 h; (k) 18, EDCl, HOBt, CH2Cl2, 25 ꢀC, 20 h, 12% for two steps; (l) Ba(OH)2‧8H2O,
THF/H2O (4/1), 0 to 25 ꢀC, 20 h, 40%; (m) 80% TFA in H2O, 25 ꢀC, 18 h, quantitative.
for details). When we adopted the procedure reported by Shibasaki
and co-workers (CuCl, PPh3 and DIPEA) [37], the isocyanoacetate
aldol reaction gave a 1:5 (14a:14b) mixture. To unambiguously
establish the configuration of the major aldol reaction product to be
(2S,3R), we subjected 13 to acid-catalyzed hydrolysis and subse-
quent Cbz protection. The NMR spectral data of the Cbz-protected
major aldol product 14a was identical with the major diaste-
reomer of the Sharpless aminohydroxylation reaction (Scheme 1),
confirming that the major isocyanoacetate aldol product was the
desired 1,2-syn-amino alcohol (see the Supplementary Information
for details).
3

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
Following the isocyanoacetate aldol reaction, the 1,2-syn-amino
alcohol 14 was treated with the glycosyl donor 8 [35], TESOTf, and
NIS to afford the glycosylation product in 60%. The azide reduction
of the glycosylation product by Zn powder and NH4Cl followed by a
subsequent Boc protection of the resulting amine gave the Boc-
protected amine 15 in 52% for two steps. The Cbz deprotection of
15 by Pd/C followed by reductive alkylation of the resulting amine
with 16 [38] and NaBH3CN/HOAc gave the carbamate 17 in 59% for
two steps. The Troc group of 17 was removed by treatment with Zn
powder in MeOH, and the resulting amine was acylated with the
carboxylic acid 18 [16] in the presence of EDCI and HOBt to afford
the amide 19. Treatment of 19 with Ba(OH)2‧8H2O provided the
carboxylic acid (40%). Finally, the global deprotection by aqueous
TFA successfully completed the synthesis of the cyclopentane-
based analog 20 (JH-MR-23) with a lipophilic side chain.
Staphylococcus aureus (strain SA113) and determined the minimal
inhibitory concentration (MIC) is 54 6.8 mg/mL (Fig. 2d).
3. Conclusion
MraY is an essential enzyme in the peptidoglycan biosynthesis
and a promising target for new antibiotic development. We
recently reported the crystal structures of MraY in complex with
representative members of the natural nucleoside inhibitors,
including muraymycin D2. To harness our recent findings and to
improve the chemical tractability of muraymycin analogs, we pre-
pared cyclopentane-based muraymycin analogs by replacing the
ribose and lipophilic peptide chain groups of muraymycins with a
cyclopentane ring and a modified lipophilic side chain. We also
explored the diastereoselective isocyanoacetate aldol reaction for
the installation of the 1,2-syn-amino alcohol group of mur-
aymycins. We found that our cyclopentane analogs are less potent
than muraymycin D2 (1d) in inhibiting MraY activity. Future
structural and functional studies are necessary to elucidate the
reason for the low efficacy of cyclopentane analogs in MraY func-
tion. However, among the cyclopentane analogs, analog 20 (JH-MR-
23) has the most potent MraY inhibition and exhibits antibacterial
activity against the Gram-positive bacteria S. aureus to levels
comparable to some of the reported muramycin analogs [18]. Our
SAR analysis of the analogs suggested that a lipophilic side chain is
important for MraY inhibition and antibacterial efficacy. Our recent
structural and functional analysis revealed that MraY contains six
druggable hot spots, all of which can be exploited in a combina-
torial manner to improve existing MraY inhibitors or develop new
types of MraY inhibitors [6]. Because our cyclopentane-based
muraymycin analogs are more amenable to modification with
2.2. Biological characterization
After the completion of analog synthesis, we performed the
UMP-Glo™ glycosyltransferase assay [39] to assess the effect of the
cyclopentane core on MraY inhibition. The analogs (10, 11 and 20)
we prepared inhibited MraY activity in a dose-dependent manner
(Fig. 2aec). Among the analogs tested, the analog 20 (JH-MR-23)
showed the most potent inhibitory activity (IC50: 75
9 mM)
against MraY from Aquifex aeolicus (MraYAA). The analogs 10 (JH-
MR-21) and 11 (JH-MR-22) without a lipophilic side chain exhibited
significantly lower inhibitory activity (IC50: 340 42
mM for 10 and
500 69 M for 11) than 20. This data indicated that a lipophilic
m
side chain is crucial for MraY inhibition. However, none of them
was as potent as muraymycin D2 [8]. We then tested the biological
activity of our most potent analog 20 (JH-MR-23) against
Fig. 2. Dose-response curves and IC50 values of (a) 10 (JH-MR-21), (b) 11 (JH-MR-22), and (c) 20 (JH-MR-23) with MraYAA solubilized in the detergent CHAPS. Each IC50 mea-
surement was made by using the UMP-Glo™ assay. Data are shown as the mean standard deviation of three technical replicates. (d) MIC of 20 (JH-MR-23) against S. aureus (strain
SA113) is 54 6.8
value is reported as mean standard error).
m
g/mL. Representative images of the MIC determination for 20 (JH-MR-23) with growth and sterility controls (4 wells each) as indicated in row B and C (n ¼ 8, MIC
4

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
various substituents, our synthetic route will be a valuable foun-
dation for the future development of new muraymycin-derived
antibiotics targeting MraY.
solid: 1H NMR (400 MHz, CDCl3)
d 3.58e3.46 (m, 3H), 2.75 (br s,
2H), 2.40e2.30 (m, 1H), 1.99e1.92 (m, 1H), 1.80e1.69 (m, 3H),
1.50e1.45 (m, 1H), 1.38e1.28 (m, 1H); HRMS (ESI) m/z 116.1073
[(MþH)þ calcd for C6H13NO 116.1069].
4. Materials and methods
1-((1S,3R)-3-(Hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,
3H)-dione (5). [Coupling] To a solution of 4 (4.50 g, 39.1 mmol) in
anhydrous DMF (135 mL) were slowly added 4 Å molecular sieves
and 3-ethoxyacryloyl isocyanate [40] (84.5 mL, 50.8 mmol) in
anhydrous benzene at ꢁ20 ꢀC. After stirring at 25 ꢀC for 15 h, the
molecular sieves were filtered off and the filtrate was concen-
trated in vacuo. The residue was purified by column chroma-
tography (silica gel, CH2Cl2/MeOH, 20/1) to afford the urea
intermediate (4.89 g, 49%) as a white solid: 1H NMR (400 MHz,
4.1. Synthesis of cyclopentane-based muraymycin analogs
General chemistry procedures. All reactions were conducted in
oven-dried glassware under nitrogen or argon. Unless otherwise
stated all reagents were purchased from commercial suppliers and
used without further purification. All solvents were American
Chemical Society (ACS) grade or better and used without further
purification except tetrahydrofuran (THF), which was freshly
distilled from sodium/benzophenone each time before use.
Analytical thin layer chromatography (TLC) was performed with
glass backed silica gel (60 Å) plates with fluorescent indication
(Whatman). Visualization was accomplished by UV irradiation at
254 nm and/or by staining with p-anisaldehyde solution. Flash
column chromatography was performed by using silica gel (particle
size 230e400 mesh, 60 Å). All 1H spectra were recorded with a
Varian 400 (400 MHz) and a Bruker 500 (500 MHz) spectrometer.
CDCl3)
d
8.63 (d, J ¼ 7.3 Hz, 1H), 8.41 (s, 1H), 7.62 (d, J ¼ 12.1 Hz,
1H), 5.24 (d, J ¼ 12.2 Hz, 1H), 4.21e4.14 (m, 1H), 3.96 (q,
J ¼ 7.1 Hz, 2H), 3.58 (d, J ¼ 5.8 Hz, 2H), 2.28e2.12 (m, 2H),
2.09e1.90 (m, 1H), 1.85e1.75 (m, 1H), 1.65e1.55 (m, 2H),
1.52e1.41 (m, 1H), 1.35 (t, J ¼ 7.0 Hz, 3H); [Uracil Formation] The
urea (4.50 g, 17.56 mmol) was dissolved in 1 N H2SO4 (150 mL)
and the resulting reaction mixture was refluxed under N2 at-
mosphere. After stirring for 30 min, the reaction was quenched
by an addition of saturated aqueous 2 N NaOH, and the resulting
mixture was diluted with EtOAc. The layers were separated, and
the aqueous layer was extracted with EtOAc. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, CH2Cl2/MeOH, 40/1) to afford
the uracil alcohol 5 (2.50 g, 68%) as a colorless oil: 1H NMR
All NMR
d values are given in parts per million (ppm) and are
referenced to the residual solvent signals (CDCl3:
d
¼ 7.26 ppm,
CD3OD:
d
¼ 3.31 ppm, (CD3)2SO:
d
¼ 2.50 ppm) for 1H NMR spectra,
or the solvent signals for 13C spectra. Coupling constants (J) are
given in Hertz (Hz) and multiplicities are indicated using the con-
ventional abbreviation (s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet or overlap of non-equivalent resonances,
br ¼ broad). Electrospray ionization (ESI) mass spectrometry (MS)
was recorded with an Agilent 1100 series (LC/MSD trap) spec-
trometer in order to obtain the molecular masses of compounds.
(400 MHz, (CD3)2SO)
d
11.20 (br s, 1H), 7.69 (d, J ¼ 8.0 Hz, 1H),
5.56 (d, J ¼ 8.0 Hz, 1H), 4.77e4.64 (m, 1H), 4.56 (br s, 1H), 3.36 (d,
J ¼ 6.2 Hz, 2H), 2.08e1.92 (m, 2H), 1.90e1.78 (m, 1H), 1.70e1.60
(m, 2H), 1.55e1.43 (m, 1H) 1.38e1.27 (m, 1H); HRMS (ESI) m/z
211.1082 [(MþH)þ calcd for C10H14N2O3 211.1077].
tert-Butyl
(1S,4R)-3-oxo-2-azabicyclo[2.2.1]heptane-2-
carboxylate (3). [Reduction] To a solution of the commercially
available (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one (2) (10 g,
91.70 mmol) in anhydrous MeOH (180 mL) was added 10% palla-
dium on activated carbon (3 g). After stirring at 25 ꢀC for 5 h under
H2 atmosphere, the reaction mixture was filtered through a pad of
Celite, and the filtrate was concentrated in vacuo to give the cor-
responding lactam [30] (9.17 g). The crude lactam was used in the
following step without further purification: 1H NMR (400 MHz,
tert-Butyl
(E)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)cyclopentyl)acrylate (6). [Oxidation] To a solution of 5
(2.50 g, 11.89 mmol) in anhydrous MeCN (400 mL) was added IBX
(8.32 g, 29.72 mmol). After stirring at 80 ꢀC for 1.5 h, the insoluble
was filtered off and the filtrate was concentrated in vacuo to afford
the corresponding aldehyde (2.50 g). The crude aldehyde was used
in the following step without further purification: 1H NMR
(400 MHz, (CD3)2CO)
d
9.98 (br s,1H), 9.71 (s,1H), 7.63 (d, J ¼ 8.0 Hz,
CDCl3)
d
5.75 (br s, 1H), 3.89 (s, 1H), 2.74 (s, 1H), 1.94e1.91 (m, 1H),
1H), 5.60 (d, J ¼ 8.0 Hz, 1H), 5.08e4.90 (m, 1H), 3.05e2.95 (m, 1H),
2.29e2.21 (m, 2H), 2.18e2.08 (m, 2H), 1.95e1.89 (m, 1H), 1.79e1.69
(m, 1H); [Wittig Reaction] To a cooled (0 ꢀC) solution of the alde-
hyde (300 mg, 1.44 mmol) in anhydrous THF (15 mL) was added
(tert-butoxycarbonylmethylene)triphenylphosphorane (2.10 g,
5.76 mmol). After stirring at 0 ꢀC for 12 h, the solvents were
removed in vacuo and the residue was purified by column chro-
1.88e1.80 (m, 2H), 1.65e1.57 (m, 2H), 1.42e1.36 (m, 1H); [Boc
Protection] A mixture of the lactam (9.17 g, 82.51 mmol), Boc2O
(28.60 g, 131.04 mmol), and DMAP (5.04 g, 41.25 mmol) in anhy-
drous MeCN (300 mL) was stirred at 25 ꢀC for 1 h. The solvents were
removed in vacuo and the residue was purified by column chro-
matography (silica gel, hexanes/EtOAc, 5/2) to afford the known
Boc-protected lactam 3 [30] (15.68 g, 81% for two steps) as a white
matography (silica gel, hexanes/i-PrOH, 6/1) to afford the
saturated ester 6 (198.33 mg, 45% for two steps) as a white solid: 1H
NMR (400 MHz, CDCl3)
a,b-un-
solid: 1H NMR (400 MHz, CDCl3)
1.95e1.90 (m, 2H),1.80e1.73 (m, 2H),1.51 (s, 9H),1.43e1.39 (m, 2H).
((1R,3S)-3-Aminocyclopentyl)methanol (4). mixture of
d 4.53 (s, 1H), 2.85 (s, 1H),
d
9.83 (s, 1H), 7.23 (d, J ¼ 8.1 Hz, 1H), 6.84
A
3
(dd, J ¼ 7.3, 1.4 Hz, 1H), 5.76 (s, 1H), 5.74 (d, J ¼ 6.6 Hz, 1H),
5.04e4.90 (m,1H), 2.82e2.62 (m,1H), 2.28e2.23 (m,1H), 2.20e2.13
(m, 1H), 2.04e1.91 (m, 1H), 1.80e1.64 (m, 2H), 1.55e1.50 (m, 1H),
(15.68 g, 74.25 mmol) and NaBH4 (5.62 g, 14.85 mmol) in MeOH
(300 mL) was stirred at 25 ꢀC for 3 h. The solvents were removed in
vacuo, and the residue was partitioned between EtOAc and H2O.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, hexanes/EtOAc, 2/1) to afford
the Boc-protected amino alcohol (12.79 g, 80%) as a white solid: 1H
1.46 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 165.79, 163.09, 151.00,
148.78, 140.65, 122.65, 102.89, 80.50, 56.06, 40.38, 37.74, 30.11,
28.13.
tert-Butyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((1R,3S)-3-
(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-3-hydroxy
NMR (400 MHz, CDCl3)
d
3.90 (br s, 1H), 3.56 (d, J ¼ 5.0 Hz, 2H),
propanoate (7). tert-Butyl hypochlorite (446 mL, 3.95 mmol) was
2.19e2.10 (m, 2H), 1.92e1.83 (m, 1H), 1.74e1.71 (m, 1H), 1.47e1.42
(m, 2H), 1.40 (s, 9H), 1.14e1.10 (m, 1H); [Boc Deprotection] A solu-
tion of the Boc-protected amino alcohol (12.79 g, 59.44 mmol) in
H2O (700 mL) was stirred at 100 ꢀC for 20 h. The solvents were
removed to give the amino alcohol 4 [30] (5.47 g, 80%) as a white
added to a solution of benzyl carbamate (92.40 mg, 3.91 mmol) in
0.6 N NaOH/n-PrOH (1/1, 15 mL) at 15 ꢀC. After stirring at 15 ꢀC for
15 min, the reaction mixture was warmed to 25 ꢀC and sequentially
treated with (DHQD)2AQN (167.10 mg, 0.19 mmol) in n-PrOH
(2.50 mL), 6 (200 mg, 0.65 mmol) in n-PrOH (2.5 mL), and
5

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
K2OsO2(OH)4 (71.80 mg, 0.19 mmol) in n-PrOH (2.50 mL). After stir-
ring at 25 ꢀC for 2 h, the reaction was quenched by an addition of H2O
and the resulting mixture was diluted with EtOAc. The layers were
separated, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with brine, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, CH2Cl2/MeOH, 50/1) to afford the
amino alcohol 7 (140 mg, 46%) as a yellow solid: 1H NMR (400 MHz,
injection: 1 mL/min) to afford 10 (1 mg, 41%) and 11 (2 mg, 91%): For
10, tR 3.263 min; HRMS (ESI) m/z 605.2816 [(MþH)þ calcd for
C
29H40N4O10 605.2711]; For 11, tR 3.329 min; 1H NMR (500 MHz,
CD3OD)
d
7.70 (d, J ¼ 7.9 Hz, 1H), 7.40e7.25 (m, 5H), 5.69 (d,
J ¼ 7.9 Hz, 1H), 5.50 (s, 2H), 5.12 (d, J ¼ 9.8 Hz, 1H), 5.03 (d, J ¼ 9.8 Hz,
1H), 4.96 (s, 1H), 4.18 (s, 1H), 4.11e4.03 (m, 2H), 4.02e3.95 (m, 2H),
3.96e3.90 (m, 1H), 3.14e3.08 (m, 2H), 2.18e2.11 (m, 2H), 2.10e2.00
(m, 1H), 1.90e1.80 (m, 1H), 1.78e1.70 (m, 1H), 1.68e1.58 (m, 2H); 13
C
CDCl3)
d
8.17 (s, 1H), 7.38e7.26 (m, 6H), 5.72 (d, J ¼ 8.4 Hz, 1H),
NMR (125 MHz, CD3OD) d 164.85, 157.75, 157.52, 151.56, 151.40,
5.52e5.44 (m,1H), 5.13 (s, 2H), 4.87e4.78 (m,1H), 4.34e4.27 (m,1H),
3.94e3.87 (m, 1H), 2.32e2.19 (m, 2H), 2.18e2.08 (m, 2H), 1.75e1.68
(m, 1H), 1.47 (s, 10H), 1.28e1.19 (m, 1H); 13C NMR (125 MHz, CDCl3)
142.87, 128.11, 127.80, 127.72, 127.68, 110.36, 101.25, 85.71, 80.86,
78.42, 75.13, 72.05, 66.61, 56.91, 56.74, 56.40, 42.71, 41.07, 34.87,
28.73, 26.22, 22.80; HRMS (ESI) m/z 549.2188 [(MþH)þ calcd for
d
170.39, 163.83, 157.16, 156.76, 151.16, 141.51, 136.26, 136.18, 102.40,
C25H32N4O10 549.2191].
82.72, 67.11, 66.96, 60.45, 57.90, 56.83, 40.88, 34.61, 29.90, 26.95,
21.06,14.19; HRMS (ESI) m/z 474.2236 [(MþH)þ calcd for C24H31N3O7
474.2235].
Methyl 5-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
cyclopentyl)-4,5-dihydrooxazole-4-carboxylate (13). [Oxidation] To
a solution of 5 (2.50 g, 11.89 mmol) in anhydrous MeCN (400 mL)
was added IBX (8.32 g, 29.72 mmol). After stirring at 80 ꢀC for 1 h,
the insoluble was filtered off and the filtrate was concentrated in
vacuo. The residue was purified by column chromatography (silica
gel, CH2Cl2/MeOH, 30/1) to afford the corresponding aldehyde
(1.72 g, 70%); [Aldol Reaction] The pre-catalyst 12 [36] (28.10 mg,
0.04 mmol) was dissolved in EtOAc (0.60 mL) and Ag2O (5.57 mg,
0.02 mmol) was added at ꢁ78 ꢀC. The resulting mixture was stirred
for approximately 1 min before sequentially treated with methyl
tert-Butyl
(2S,3R)-3-(((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-
diethyltetrahydrofuro[3,4-d] [1,3]dioxol-4-yl)oxy)-2-(((benzyloxy)
carbonyl)amino)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)cyclopentyl)propanoate (9). [Glycosylation] To a solution of 7
(90 mg, 0.19 mmol) in CH2Cl2 (8 mL) were added 8 [35] (78.70 mg,
0.25 mmol), 4 Å molecular sieves, and NIS (74.80 mg, 0.33 mmol).
Afterstirringat25ꢀC for10min, TESOTf(20
mL,0.09mmol)wasadded
and the resulting mixture was stirred for 30 min. The reaction was
quenched by an addition of saturated aqueous NaHCO3, and the
resulting mixture was diluted with EtOAc. The layers were separated,
and the aqueous layer was extracted with EtOAc. The combined
organic layers were washed with brine, dried overanhydrous Na2SO4,
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH, 40/1) to afford the corre-
isocyanoacetate (51.50 mL, 0.56 mmol) and powdered 4 Å molecular
sieves at ꢁ78 ꢀC. The aldehyde (200 mg, 0.96 mmol) in EtOAc
(1 mL) was added to the reaction mixture. The resulting mixture
was slowly warmed to 0 ꢀC and stirred at 0 ꢀC for 1.5 d Ag2O was
removed by filtering through a pad of Celite with MeOH to afford
the trans-oxazoline 13. The crude 13 was used in next step without
further purification.
1
sponding azide (60 mg, 53%) as a yellow solid: H NMR (400 MHz,
CDCl3)
d
9.20 (br s,1H), 7.41e7.26 (m, 5H), 7.22 (d, J ¼ 7.7 Hz,1H), 6.06
Methyl
(2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((1R,3S)-3-
(d, J ¼ 9.6 Hz, 1H), 5.72 (d, J ¼ 8.2 Hz, 1H), 5.11 (s, 1H), 5.09 (d,
J ¼ 13.9 Hz, 2H), 4.91e4.77 (m, 1H), 4.58e4.52 (m, 2H), 4.30 (d,
J ¼ 9.8 Hz, 1H), 4.27e4.21 (m, 1H), 4.09 (q, J ¼ 7.2 Hz, 2H), 4.00 (d,
J ¼ 7.2 Hz, 1H), 3.50e3.32 (m, 2H), 2.30e2.17 (m, 5H), 2.00e1.89 (m,
2H),1.70e1.60 (m, 2H),1.58e1.48 (m, 2H),1.45 (s, 9H), 0.90e0.78 (m,
(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-3-hydroxy
propanoate (14a). [Hydrolysis] To a solution of 13 (200 mg,
0.65mmol)inTHF(1.40mL)wasadded4NHClindioxane(0.70mL)at
0 ꢀC. After stirring under N2 at 25 ꢀC for 1 h, the reaction mixture was
concentrated in vacuo to afford the crude amino alcohol, which was
used in the following step without further purification; [Cbz Protec-
tion] To a solution of the crude amino alcohol (200 mg, 0.67 mmol) in
THF/H2O (2/1, 6 mL) were added NaHCO3 (113 mg, 1.35 mmol) and
CbzCl (0.14 mL,1.01 mmol). After stirring at 0 ꢀC for 15 h, the reaction
was quenched by an addition of H2O, and the resulting mixture was
diluted with EtOAc. The layers were separated, and the aqueous layer
was extracted with EtOAc. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography (silica gel,
CH2Cl2/MeOH, 30/1) to afford the syn-amino alcohol 14a (84.9 mg,
20.5% for three steps) and the diastereomeric syn-amino alcohol 14b
(42.7 mg, 10.3% for three steps) each as a white solid: Data for the
6H); 13C NMR (125 MHz, CDCl3)
d 178.07,171.12,169.29,156.88,151.07,
141.02, 136.50, 117.42, 111.13, 102.54, 86.28, 84.66, 84.22, 82.54, 81.94,
66.97, 60.37, 56.52, 56.21, 53.19, 40.94, 35.55, 29.58, 29.45, 28.87,
27.94, 21.01, 14.17, 8.35, 7.35, 6.77, 6.39; HRMS (ESI) m/z 721.3161
[(MþNa)þ calcd for C34H46N6O10 721.3168]; [Azide Reduction] To a
solution of the azide (5 mg, 0.007 mmol) in H2O (0.05 mL) and THF/
toluene (1/1, 1 mL) was added PPh3 (3.70 mg, 0.01 mmol). After stir-
ring at 25 ꢀC for 12 h, the solvents were removed in vacuo and the
residue was purified by column chromatography (silica gel, CH2Cl2/
MeOH, 10/1) to afford the amine 9 (3 mg, 63%) as a white solid: 1H
NMR (400 MHz, CDCl3)
d
7.38e7.24 (m, 6H), 5.71 (d, J ¼ 8.2 Hz, 1H),
5.14e5.09 (m, 3H), 4.84e4.78 (m, 1H), 4.62e4.49 (m 3H), 4.35 (d,
J ¼ 9.4 Hz, 1H), 4.24e4.17 (m, 1H), 4.03 (d, J ¼ 8.4 Hz, 1H), 2.90e2.84
(m, 1H), 2.72e2.68 (m, 1H), 2.58e2.50 (m, 1H), 1.69e1.62 (m, 6H),
1.57e1.51 (m, 5H), 1.45 (s, 9H), 0.90e0.79 (m, 6H); HRMS (ESI) m/z
673.3444 [(MþH)þ calcd for C34H48N4O10 673.3443].
(2S,3R)-syn-amino alcohol: 1H NMR (400 MHz, CDCl3)
d 7.43e7.28 (m,
5H), 7.23 (d, J ¼ 7.8 Hz, 1H), 5.76 (d, J ¼ 9.5 Hz, 1H, eNH), 5.70 (d,
J ¼ 8.0 Hz,1H), 5.12 (s, 2H), 4.82e4.74 (m,1H), 4.47 (d, J ¼ 9.7 Hz,1H),
4.01 (d, J ¼ 8.4 Hz,1H), 3.76 (s, 3H), 2.29e2.20 (m, 1H), 2.18e2.05 (m,
2H), 1.94e1.85 (m, 1H), 1.78e1.70 (m, 1H), 1.68e1.57 (m, 2H); HRMS
(ESI) m/z 432.1771 [(MþH)þ calcd for C21H25N3O7 432.1765]; Data for
the diastereomeric (2R,3S)-syn-amino alcohol: 1H NMR (400 MHz,
tert-Butyl
(2S,3R)-3-(((2R,3R,4S,5R)-5-(aminomethyl)-3,4-dihy
droxytetrahydrofuran-2-yl)oxy)-2-(((benzyloxy)carbonyl)amino)-3-
((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)
propanoate (10). Compound 9 (3 mg, 0.004 mmol) was treated with
80% TFA in THF/H2O (1/1, 1 mL) and stirred at 25 ꢀC for 14 h. The
reaction mixture was concentrated in vacuo and triturated from
CH2Cl2 to afford the partially deprotected t-Bu ester 10 (JH-MR-21)
and the fully deprotected carboxylic acid 11 (JH-MR-22). The global
deprotection reaction 10 or 11 as the final product depending on
purity of 9 and reaction time. Compounds 10 and 11 were purified by
HPLC (YMC J’sphere ODS M80, 10 mm ꢂ 150 mm, 0.1% formic acid, a
linear gradient from 60 to 99% of MeCNeH2O for 30 min, flow
CDCl3)
d
8.80 (br s,1H), 7.42e7.28 (m, 5H), 7.20 (d, J ¼ 8.0 Hz,1H), 5.74
(d, J ¼ 7.5 Hz,1H), 5.46 (dd, J ¼ 11.8, 9.8 Hz, 2H), 5.14 (s, 2H), 4.89e4.86
(m, 1H), 4.66 (d, J ¼ 9.8 Hz, 1H), 3.74 (s, 3H), 3.67e3.60 (m, 1H),
2.41e2.30(m,1H), 2.21e2.07(m, 2H),1.74e1.70(m,1H),1.68e1.64(m,
1H), 1.50e1.46 (m, 2H).
Methyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-(((3aR,4R,6-
R,6aR)-6-(((tert-butoxycarbonyl)amino)methyl)-2,2-diethyltetrahy
drofuro[3,4-d] [1,3]dioxol-4-yl)oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)cyclopentyl)propanoate
(15).
6

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
[Glycosylation] To a solution of 14 (20 mg, 0.04 mmol) in CH2Cl2
(1.50 mL) were added 8 [35] (20 mg, 0.06 mmol), 4 Å molecular
sieves, and NIS (18 mg, 0.08 mmol). After stirring at 25 ꢀC for
through a pad of Celite, and the filtrate was concentrated in vacuo
to give the corresponding amine. The crude amine was used in the
following step without further purification; HRMS (ESI) m/z
597.3138 [(MþH)þ calcd for C28H44N4O10 597.3130]; [Reductive
amination] To a solution of the crude amine (45 mg, 0.05 mmol) in
anhydrous MeOH (3 mL) were treated with 16 [38] (22.60 mg,
10 min, TESOTf (5
mL, 0.02 mmol) was added and the resulting
solution was stirred for 30 min. The reaction was quenched by an
addition of saturated aqueous NaHCO3, and the resulting mixture
was diluted with EtOAc. The layers were separated, and the
aqueous layer was extracted with EtOAc. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by column chro-
matography (silica gel, CH2Cl2/MeOH, 30/1) to afford the corre-
sponding azide (15.70 mg, 60%) as a white solid: 1H NMR (400 MHz,
0.09 mmol) in HOAc (30 mL, 0.54 mmol) and NaBH3CN (15 mg,
0.24 mmol). After stirring at 25 ꢀC for 15 h, the reaction was
quenched by an addition of saturated aqueous NaHCO3, and the
resulting mixture was diluted with EtOAc. The layers were sepa-
rated, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, CH2Cl2/MeOH, 30/
1) to afford the carbamate 17 (27 mg, 59% for two steps) as a white
CDCl3)
d
8.80 (br s, 1H), 7.42e7.29 (m, 5H), 7.22 (d, J ¼ 8.1 Hz, 1H),
6.05 (d, J ¼ 9.9 Hz, 1H), 5.74 (dd, J ¼ 8.0, 2.4 Hz, 1H), 5.14 (d,
J ¼ 3.4 Hz, 2H), 5.08 (s, 1H), 4.88e4.85 (m, 1H), 4.60 (dd, J ¼ 5.9,
1.6 Hz,1H), 4.54 (d, J ¼ 6.1 Hz,1H), 4.47 (d, J ¼ 9.7 Hz,1H), 4.28e4.23
(m, 1H), 4.07 (d, J ¼ 8.3 Hz, 1H), 3.75 (s, 3H), 3.48e3.40 (m, 2H),
2.33e2.25 (m, 2H), 2.20e2.14 (m, 1H), 2.01e1.91 (m, 2H), 1.80e1.70
(m, 2H), 1.69e1.63 (m, 2H), 1.60e1.50 (m, 2H), 0.98e0.78 (m, 6H);
HRMS (ESI) m/z 657.2879 [(MþH)þ calcd for C31H40N6O10
657.2883]; [Azide Reduction] To a solution of the azide (19 mg,
0.02 mmol) in EtOH/H2O (3/1, 0.80 mL) were added activated Zn
powder (2.60 mg, 0.03 mmol) and NH4Cl (3.70 mg, 0.07 mmol).
After stirring at 25 ꢀC for 1.5 h, the reaction was quenched by an
addition of saturated aqueous NaHCO3, and the resulting mixture
was diluted with CH2Cl2. The layers were separated, and the
aqueous layer was extracted with CH2Cl2. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo to afford the corresponding amine. The crude
amine was used in the following step without further purification:
solid: 1H NMR (400 MHz, CDCl3)
d 8.89 (br s, 1H), 7.25e7.22 (m,
1H), 5.87e5.76 (m, 1H), 5.73 (d, J ¼ 8.1 Hz, 1H), 5.06 (s, 1H),
4.88e4.79 (m, 1H), 4.72e4.70 (m, 2H), 4.60 (d, J ¼ 6.0 Hz, 1H),
4.50e4.46 (m, 1H), 4.29e4.24 (m, 1H), 3.75 (s, 3H), 3.36e3.29 (m,
3H), 3.26e3.16 (m, 4H), 2.93e2.82 (m, 1H), 2.49e2.32 (m, 2H),
2.31e2.23 (m, 1H), 2.21e2.12 (m, 1H), 1.84e1.80 (m, 2H), 1.66e1.49
(m, 4H), 1.48e1.43 (m, 3H), 1.42 (s, 12H), 0.86e0.84 (m, 6H); 13C
NMR (125 MHz, CDCl3)
d 178.02, 173.79, 163.00, 154.58, 150.81,
141.21, 116.90, 112.71, 103.69, 102.56, 86.96, 85.51, 82.32, 79.22,
74.46, 74.39, 64.08, 56.73, 53.43, 52.36, 46.54, 43.17, 41.04, 39.80,
37.22, 35.01, 32.21, 29.90, 29.82, 29.42, 29.04, 28.99, 27.08, 8.41,
7.52; HRMS (ESI) m/z 828.2740 [(MþH)þ calcd for C34H52Cl3N5O12
828.2751].
Methyl
(2S,3R)-3-(((3aR,4R,6R,6aR)-6-(((tert-butoxycarbonyl)
[1,3]dioxol-4-yl)
amino)methyl)-2,2-diethyltetrahydrofuro[3,4-d]
1H NMR (400 MHz, CDCl3)
d
7.51 (br s, 1H), 7.40e7.29 (m, 5H),
oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclo-
pentyl)-2-((3-((S)-2-heptadecanamido-5-(3-((2,2,5,6,8-pentamethyl
chroman-7-yl)sulfonyl)guanidino)pentanamido)propyl)amino)pro
panoate (19). [Troc Deprotection] A solution of 17 (27 mg, 0.03 mmol)
in anhydrous MeOH (1.50 mL) were treated with NH4Cl (49 mg,
0.92 mmol) and Zn powder (97% purity, 31.80 mg, 0.48 mmol). After
stirring at 25 ꢀC for 25 h, the insoluble was filtered through a pad of
Celite, and the filtrate was concentrated in vacuo to give the corre-
sponding amine. The crude amine was used in the following step
withoutfurtherpurification;HRMS (ESI) m/z 654.3713 [(MþH)þ calcd
for C31H51N5O10 654.3709]; [Coupling] To a solution of the crude
amine (27 mg, 0.04) in CH2Cl2 (1 mL) were added EDCI (3.20 mg,
0.01 mmol), HOBt (1.9 mg, 0.01 mmol) and 18 [16] (8.40 mg,
0.01 mmol). After stirring at 25 ꢀC for 20 h, the reactionwas quenched
by an addition of 1 N HCl solution, and the reaction mixture was
diluted with EtOAc. The layers were separated, and the aqueous layer
was extracted with EtOAc. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The combined organic layers were purified by column chromatog-
raphy (silica gel, CH2Cl2/MeOH, 30/1)toaffordtheamide19 (5mg,12%
7.25e7.21 (d, J ¼ 8.2 Hz,1H), 5.75 (d, J ¼ 8.0 Hz,1H), 5.12 (s, 2H), 5.09
(s, 1H), 4.91e4.82 (m, 1H), 4.60e4.50 (m, 3H), 4.25 (br s, 1H), 4.08
(d, J ¼ 7.6 Hz, 1H), 3.77 (s, 3H), 2.97e2.92 (m, 1H), 2.87e2.82 (m,
1H), 2.28e2.21 (m, 3H), 2.15e2.10 (m, 1H), 2.05e1.90 (m, 1H),
1.75e1.69 (m, 2H), 1.68e1.64 (m, 2H), 1.60e1.50 (m, 2H), 0.92e0.82
(m, 6H); [Boc Protection] To a solution of the crude amine (8 mg,
0.01 mmol) in anhydrous CH2Cl2 (1 mL) were added NaHCO3 (2 mg,
0.02 mmol) and Boc2O (11 mg, 0.04 mmol). After stirring at 25 ꢀC
for 2 h, the reaction was quenched by an addition of H2O, and the
resulting mixture was diluted with CH2Cl2. The layers were sepa-
rated, and the aqueous layer was extracted with CH2Cl2. The residue
was purified by column chromatography (silica gel, CH2Cl2/MeOH,
20/1) to afford the Boc-protected glycosylation product 15
(9.20 mg, 52% for two steps) as a white solid: 1H NMR (400 MHz,
CDCl3)
d
8.34 (s, 1H), 7.40e7.29 (m, 5H), 7.23 (d, J ¼ 8.9 Hz, 1H), 5.75
(dd, J ¼ 5.8, 2.2 Hz, 1H), 5.58e5.45 (m, 1H), 5.40 (br s, 1H), 5.12 (d,
J ¼ 7.5 Hz, 2H), 5.07 (s, 1H), 4.91e4.82 (m, 1H), 4.60 (d, J ¼ 6.2 Hz,
1H), 4.54 (d, J ¼ 9.8 Hz,1H), 4.49 (d, J ¼ 6.1 Hz,1H), 4.25 (t, J ¼ 5.1 Hz,
1H), 4.04 (d, J ¼ 8.0 Hz, 1H), 3.79 (s, 3H), 3.25e3.18 (m, 2H),
2.28e2.21 (m, 2H), 2.00e1.88 (m, 1H), 1.75e1.62 (m, 6H), 1.60e1.50
for two steps) as a white solid: 1H NMR (400 MHz, CD3OD)
d 7.67 (d,
(m, 11H), 0.92e0.82 (m, 6H); 13C NMR (125 MHz, CDCl3)
d
171.43,
J ¼ 8.0 Hz,1H), 5.67 (d, J ¼ 7.9 Hz,1H), 5.08 (s,1H), 4.64e4.54 (m, 2H),
4.33e4.22 (m,1H), 4.18e4.10 (m,1H), 3.92e3.87 (m,1H), 3.73 (s, 3H),
3.40e3.20 (m, 3H), 3.18e3.09 (m, 2H), 2.75e2.72 (m, 1H), 2.68e2.62
(m, 2H), 2.55e2.54 (m, 6H), 2.49e2.39 (m, 2H), 2.23e2.18 (m, 3H),
2.11e2.06 (m, 4H),1.99 (s,1H),1.90e1.47 (m, 25H),1.42 (s, 3H),1.29 (s,
3H),1.27(s, 9H),1.24e1.12(m,21H),0.88e0.86(m, 9H);HRMS(ESI)m/
z 1328.8127 [(MþH)þ calcd for C68H113N9O15S 1328.8150].
163.96, 156.52, 156.01, 150.82, 141.03, 136.00, 128.31, 128.25, 117.17,
112.51, 106.68, 87.00, 86.31, 85.24, 82.21, 79.52, 67.41, 56.69, 56.39,
52.94, 43.06, 41.00, 35.28, 29.64, 29.29, 29.00, 26.87, 8.40, 7.55;
HRMS (ESI) m/z 753.3324 [(MþNa)þ calcd for C36H50N4O12
753.3317].
Methyl (2S,3R)-3-(((3aR,4R,6R,6aR)-6-(((tert-butoxycarbonyl)
amino)methyl)-2,2-diethyltetrahydrofuro[3,4-d] [1,3]dioxol-4-yl)
oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyc
lopentyl)-2-((3-(((2,2,2-trichloroethoxy)carbonyl)amino)propyl)
amino)propanoate (17). [Cbz Deprotection] To a solution of 15
(45 mg, 0.06 mmol) in anhydrous MeOH (2.25 mL) was added 10%
palladium on activated carbon (27 mg). After stirring under H2
atmosphere at 25 ꢀC for 3 h, the reaction mixture was filtered
Methyl
(2S,3R)-3-(((2R,3R,4S,5R)-5-(aminomethyl)-3,4-
dihydroxytetrahydrofuran-2-yl)oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)cyclopentyl)-2-((3-((S)-5-guanidino-2-
heptadecanamidopentanamido)propyl)amino)propanoate formate
(20). [Hydrolysis] Ba(OH)2‧8H2O (3.20 mg, 0.01 mmol) was added
to a solution of 19 (5 mg, 0.003 mmol) in THF/H2O (4/1, 0.10 mL) at
0
ꢀC and the resulting mixture was stirred at 0 ꢀC for 10 min. The
7

S.-H. Kwak, W.Y. Lim, A. Hao et al.
European Journal of Medicinal Chemistry 215 (2021) 113272
mixture was warmed to 25 ꢀC and stirred for 20 h. The reaction was
quenched by an addition of 1 N HCl and diluted with EtOAc. The
layers were separated, and the aqueous layer was extracted with
EtOAc. The organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by column chro-
matography (silica gel, CH2Cl2/MeOH, 5/1) to afford the corre-
sponding carboxylic acid (2 mg, 40%) as a white solid: 1H NMR
approval to the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
(500 MHz, CD3OD)
d
7.70 (d, J ¼ 8.0 Hz, 1H), 5.69 (d, J ¼ 7.9 Hz, 1H),
5.18 (s, 1H), 4.70e4.58 (m, 2H), 4.30e4.25 (m, 1H), 4.22e4.18 (m,
1H), 3.82 (br s,1H), 3.22e3.18 (m, 3H), 3.13e3.08 (m, 2H), 2.71e2.68
(m, 3H), 2.60e2.57 (m, 6H), 2.42e2.37 (m, 2H), 2.23e2.18 (m, 3H),
2.10 (s, 2H), 2.01 (s, 1H),1.88e1.53 (m, 25H), 1.42 (s, 3H), 1.31 (s, 3H),
1.28 (s, 9H), 1.26e1.20 (m, 21H), 0.88e0.86 (m, 9H); HRMS (ESI) m/z
1314.7990 [(MþH)þ calcd for C67H111N9O15S 1314.7993]; [Global
Deprotection] After the carboxylic acid (2 mg, 0.001 mmol) was
treated with 80% TFA in H2O (0.30 mL), the resulting mixture was
stirred at 25 ꢀC for 18 h. The reaction mixture was concentrated in
vacuo and triturated from CH2Cl2 to afford 20 (2 mg, quantitative)
as a white solid: HRMS (ESI) m/z 880.5862 [(MþH)þ calcd for
Acknowledgments
This work was supported by the grant from the National Insti-
tute of General Medical Sciences (R01GM120594).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113272.
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Author contributions
S.-H.K., W.Y.L., A.H., and E.H.M. contributed equally; S.-Y.L. and
J.H. held overall responsibility for the study; All authors have given
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