DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors

Article abstract of DOI:10.1021/acs.jmedchem.0c00545

Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC50 value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the IC50 (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC50: 1.25 μM) inhibits growth of PD002 at 0.0024-0.16 μM in combination with 0.10-2.0 μM CPPB (IC50: 35 μM).

Full text of DOI:10.1021/acs.jmedchem.0c00545

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Article
DPAGT1 Inhibitors of Capuramycin Analogues
and Their Antimigratory Activities of Solid Tumors
Katsuhiko Mitachi, Rita G Kansal, Kirk E. Hevener, Cody D Gillman, Syed M Hussain, Hyun Gi
Yun, Gustavo A. Miranda-Carboni, Evan S Glazer, William M. Clemons Jr., and Michio Kurosu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00545 • Publication Date (Web): 04 Sep 2020
Downloaded from pubs.acs.org on September 4, 2020
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DPAGT1 Inhibitors of Capuramycin Analogues and
Their Antimigratory Activities of Solid Tumors
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a
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Katsuhiko Mitachi, Rita G. Kansal, Kirk E. Hevener, Cody D. Gillman, Syed M. Hussain, Hyun Gi
c
d
b
c
a
Yun, Gustavo A. Miranda-Carboni, Evan S. Glazer, William M. Clemons Jr., and Michio Kurosu* .
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science
Center, 881 Madison Avenue, Memphis, TN 38163, USA
bDepartment of Surgery and Center for Cancer Research, College of Medicine, University of Tennessee
Health Science Center, 910 Madison St., Suite 300, Memphis, TN 38163, USA
cDivision of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California
Blvd. Pasadena, CA 91125, USA
dDepartment of Medicine, Division of Hematology-Oncology, University of Tennessee Health Science
Center, 19 S. Manassas Avenue, Memphis, TN 38163, USA
KEYWORDS. Capuramycin analogues, DPAGT1 inhibitor, Antimigratory activity, Snail zinc-finger
transcription factors, E-Cadherin, Synergistic effect, Computational chemistry
ABSTRACT:
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Capuramycin displays narrow spectrum of antibacterial activity by targeting bacterial translocase I
(
MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1)
inhibitor, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits
DPAGT1 enzyme with an IC50 value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does
not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits
migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in
order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail, but
does not reduce E-cadherin expression level at the IC50 (DPAGT1) concentration. CPPB displays a strong
synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic
adenocarcinoma, PD002: paclitaxel (IC50 1.25 M) inhibits growth of PD002 at 0.0024-0.16 M in
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combination with 0.10-2.0 M of CPPB (IC50 35 M).
INTRODUCTION
Capuramycin (CAP (1), Figure 1) is a nucleoside antibiotic, which has a narrow-spectrum of activity
against Gram-positive bacteria including Mycobacterium tuberculosis (Mtb).1-3 To date, medicinal
chemistry efforts on capuramycin have been focused on developing a new TB drug; new capuramycin
analogues have been synthesized to improve bacterial phospho-MurNAc-pentapeptide translocase I (MraY
and MurX for Mycobacterium spp.) enzyme as well as antimycobacterial activities.1,4,5 Somewhat recently,
the anti-Clostridioides difficile (formerly Clostridium difficile) activity of a capuramycin analogue has been
6
7
reported. Capuramycin is a specific inhibitor of MraY with the IC50 value of 0.13 M, however, some
other nucleoside antibiotics (e.g., muraymycin A1 and tunicamycins) display activity against MraY, WecA
(polyprenyl phosphate-GlcNAc-1-phosphate transferase), and its human homologue, DPAGT1 (N-
acetylglucosaminephosphotransferase1)-type phosphotransferases.8-14 MraY is an essential enzyme for
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growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide
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(
Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I). WecA catalyzes the transformation from
UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is
responsible for the biosynthesis of mycolylarabinogalactan in Mycobacterium tuberculosis (Mtb). WecA is
an essential enzyme for the growth of Mtb and some other bacteria. Biochemical studies of WecA enzyme
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are hampered by lack of selective inhibitor molecules. Tunicamycin shows inhibitory activity against these
phosphotransferases with the IC50 values of 2.9 M (MraY/MurX), 0.15 M (WecA), and 1.5 M
(
DPAGT1).9,10 CPZEN-45, an antimycobacterial MraY inhibitor, was reported to exhibit WecA inhibitory
1
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activity (IC50 ~0.084 M). We showed that 2’O-methyl capuramycin (OM-CAP (2), formerly UT-01320,
Figure 1) does not exhibit MraY/MurX inhibitory activity, but displays a strong WecA inhibitory activity
IC50 0.060 M). In vitro cytotoxicity of tunicamycin has been documented in a number of articles.17
1
,9
(
Acute toxicity of tunicamycin due to its narrow therapeutic window (LD50: 2.0 mg/kg, LD100: 3.5 mg/kg
mice, IP) discourages scientists from developing tunicamycin for new antibacterial, antifungal, or anti-
cancer agents.18,19 A large number of scientists believe that cytotoxicity of tunicamycin is attributable to its
interaction with DPAGT1, which catalyzes the first and rate limiting step in the dolichol-linked
oligosaccharide pathway in N-linked glycoprotein biosynthesis (Figure 2).10,12 In contrast, OM-CAP (2)
possesses the same level of DPAGT1 inhibitory activity (IC50 4.5 M) to tunicamycins (IC50 1.5 M), but
it does not show cytotoxicity against healthy cells (e.g., Vero and HPNE cells) at 50 M and some cancer
cell lines (e.g., L1210, KB, AsPC-1, PANC-1, LoVo, SK-OV-3) at 10 M. A sharp difference in the
cytotoxicity profiles between tunicamycins (11, Figure 1) and OM-CAP (2) raises the question of whether
selective inhibition of DPAGT1 enzyme function in certain cells/organs by small molecules does not cause
unacceptable level of toxicity against human during chemotherapy. We have recently engineered the
structure of muraymycin to yield strong DPAGT1 inhibitors; one of these analogues, APPB (12, Figure 1),
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showed a promising antiproliferative effect on a series of solid cancer cell lines that required overexpression
of the DPAGT1 gene in their growth and cancer progression.11,13,14 The design of APPB was originated from
the discovery of DPAGT1 inhibitors of capuramycin analogues. In this article, we report structure-activity
relationship (SAR) studies of capuramycin to identify novel DPAGT1 inhibitors, and in vitro anti-invasion
and anti-metastasis activity of a new capuramycin analogue DPAGT1 inhibitor, CPPB (capuramycin
phenoxypiperidinylbenzylamide analogue, 5) (Figure 1). A unique synergistic effect was observed against
a patient-derived pancreatic adenocarcinoma, PD002 in a combination of CPPB with paclitaxel. We
demonstrated key interactions of CPPB with DPAGT1 via molecular docking studies. Lastly, we report a
semi-synthetic method to deliver enough CPPB for future in vivo studies.
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Insert Figure 1 and Figure2
RESULTS AND DISCUSSION
Chemistry and Structure activity relationship (SAR). Sankyo (currently Daiichi-Sankyo) and Sequella
have reported several capuramycin (CAP, 1) analogues that have improved MraY enzyme and bacterial
growth inhibitory activities.2,3,5,20 Their SAR studies rely on a capuramycin biosynthetic intermediate, A-
500359E, which allows delivery of novel amide molecules (R
1
group) having an ester (R ) functional group
2
(Figure 3). The capuramycin analogue, SQ992, has an interesting antibacterial characteristic with
2
1
antimycobacterial activity in vivo. We have accomplished a total chemical synthesis of capuramycin and
its analogues.22, To date, we have identified an novel analogue possessing improved MraY inhibitory
23
activity, Cap-3-amino-1,4-benzodiazepine-2-one analogue, and a selective WecA inhibitor, OM-Cap (2),
via our total synthetic approach (Figure 3).1,9 Since our first report on a total synthesis of capuramycin, a
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few improvements of the synthetic scheme have been made: we introduced acid-cleavable protecting groups
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for the uridine ureido nitrogen (3-postion in a) and primary alcohol (6”-position in c) (Scheme 1). The
MDPM and MTPM groups can simultaneously be removed with 30% TFA to form the key synthetic
intermediate A for capuramycin. In our on-going SAR of capuramycin, commercially available protecting
groups (BOM and chloroacetyl groups) for the ureido nitrogen (3-position) and C6’’-alcohol were decided
to apply in our SAR tactics. Success of hydrogenolytic cleavage of the BOM group and tolerability of the
chloroacetyl group were difficult to predict in capuramycin synthesis. To establish the new protecting group
strategy, we first demonstrated the synthesis of iso-capuramycin (I-Cap) (Scheme 2A). Syntheses of all
building blocks and the experimental procedures for their synthetic steps in Scheme 2 and 3 are summarized
in Supporting Information (SI). Highlights of syntheses of new capuramycin analogues are illustrated in
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Scheme 2 and 3. The (S)-cyanohydrin 13 was subjected to NIS-AgBF
with the thioglycoside 14 to yield the -mannosylated cyanohydrin 15 in 78% yield. The cyano group of
5 was hydrated using HgCl -aldoxime in aq. EtOH, and the BOM and chloroacetyl groups of the generated
4
promoted -selective mannosylation
2
2
1
2
amide were deprotected stepwisely: dechloroacetylation with thiourea followed by hydrogenation with 10%
i
Pd-C in AcOH- PrOH-THF provided the C6”-free alcohol 16 in 53% overall yield. Oxidation-elimination
reaction of 16 with SO
unsaturated aldehyde 17 in quantitative yield (determined by H NMR analysis). After all volatiles were
removed, the aldehyde 17 was oxidized to the corresponding carboxylic acid 18 by using NaClO in the
and 2-methyl-2-butene. The resulting carboxylic acid 18 was coupled with (2S)-
aminocaprolactam by using a standard peptide-forming reaction condition (HOBt, EDCI, and NMM) to
yield the coupling product 19 in 70% overall yield from 16. Saponification of 19 by using Et N in MeOH
provided I-Cap (3) in quantitative yield. Similarly, dimethyl-capuramycin (DM-CAP) was synthesized in
3
•pyridine in a solvent system (CH
2
Cl
2
/Et N/DMSO = 10/2/1) provided the -
3
1
2
2
4
presence of NaH
2
PO
4
3
3
1% overall yield from the cyanohydrin-acetonide 20. CAP (1) and its three analogues, OM-CAP (2), I-
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CAP (3), and DM-CAP (4) were evaluated in enzyme inhibitory assays against bacterial
phosphotransferases, MraY and WecA, and archaeal and human dolichyl-phosphate GlcNAc-1-
phosphotransferases, AglH and DPAGT1.7,9,11 CAP is a selective MraY inhibitor that does not display
inhibitory activity against WecA, AglH, and DPAGT1 (Table 1). Previously, OM-CAP (2) was identified
as a selective WecA inhibitor (IC50 0.060 M) that does not possess MraY inhibitory activity (IC50 >50 M
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(entry 2 in Table 1). In this program, it was realized that OM-CAP (2) has inhibitory activity against AglH
and DPAGT1 with the IC50 values of 2.5 and 4.5 M, respectively (entry 2). I-CAP (3) showed enzyme
inhibitory activity against MraY, WecA, AglH, and DPAGT1 with the IC50 between 8.5-30 M
concentrations (entry 3). On the contrary, DM-CAP (4) showed only a weak WecA inhibitory activity (IC50
3
5.0 M) (entry 4). In recent reports on co-crystal structures of DAPGT1 with tunicamycin (PDB: 5O5E
and 6BW6), the fatty acid chain of tunicamycin is occupied in the hydrophobic tunnel (the proposed
dolichol-phosphate (Dol-P) binding site).25,26 We speculated that introduction of pharmacologically
amenable hydrophobic groups that occupy the proposed Dol-P binding site is essential to exhibit strong
DPAGT1 inhibitory activity. In virtual screening of a hydrophobic group (excluding fatty acids) using the
structure of DPAGT1 with bound tunicamycin (PDB: 6BW6), an analogue (e.g.,
(((trifluoromethoxy)phenoxy)piperidinyl)benzylamide)
of
(((trifluoromethoxy)phenoxy)piperidin-1-
yl)phenol group (i.e. 34 in Scheme 3) found in a new anti-TB drug, delamanid,27,28 was suggested to be a
reasonable
fatty
acid
surrogate,
whose
capuramycin
derivatives
(capuramycin
phenoxypiperidinylbenzylamide (CPPB, 5), iso-capuramycin phenoxypiperidinylbenzylamide (I-CPPB, 6),
O-methyl capuramycin phenoxypiperidinylbenzylamide (OM-CPPB, 7), demethyl capuramycin
phenoxypiperidinylbenzylamide (DM-CPPB, 8), capuramycin phenoxypiperidinylbenzylamine (CPPA, 9),
and iso-capuramycin phenoxypiperidinylbenzylamine (I-CPPA, 10)) could bind to DPAGT1 with high
affinity. However, the docking program used in these studies is not able to distinguish between low- and
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high-binding molecules: all (((trifluoromethoxy)phenoxy)piperidin-1-yl)phenyl)methanamino derivatives
provided “good” GlideScores of between -12.9~-16.6 (see Table S1 in SI). Therefore, we decided to
synthesize all analogues (CPPB, I-CPPB, OM-CPPB, DM-CPPB, CPPA, and I-CPPA) identified in the
virtual screening and evaluate their phosphotransferase inhibitory activity.
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The carboxylic acid intermediate 32 for CAP (1) was subjected to peptide coupling reaction with
(((trifluoromethoxy)phenoxy)piperidin-1-yl)phenyl)methylamine (34), providing the protected CPPB, 37.
Saponification of 37 and purification by reverse HPLC yielded CPPB (5) in 95% overall yield from 37.
Similarly, I-CPPB (6), OM-CPPB (7), and DM-CPPB (8) were synthesized from the mannuronic acid
derivatives 18, 33, and 31 in 24-34% overall yield (Scheme 3A). Capuramycin
phenoxypiperidinylbenzylamine analogues, CPPA (9) and I-CPPA (10), were synthesized via reductive
aminations of the aldehydes 29 and 17 with the amine 34, furnishing the desired products in 63-65% overall
yield from 26 and 16 after saponification. MraY, WecA, AglH, and DPAGT1 enzyme inhibitory activity
assays for the six (((trifluoromethoxy)phenoxy)piperidin-1-yl)phenyl)methylamine analogues revealed that
CPPB (5) and I-CPPB (6) are high nM range DPAGT1 inhibitors (entries 5 and 6 in Table 1), however, the
O-methylation and demethylation analogues (OM-CPPB (7) and DM-CPPB (8)) turned out to be very low-
or no-DPAGT1 inhibitor (entries 7 and 8). The secondary amine analogues, CPPA (9) and I-CPPA (10), did
not inhibit all phosphotransferases tested in Table 1 at 50 M concentration (entries 9 and 10). CPPB was
determined to be three times stronger DAPGT1 inhibitor (IC50 0.20 M) than I-CPPB (entry 5 vs. 6). I-
CPPB did not display MraY inhibitory activity, but showed a very weak WecA and AglH enzyme inhibitory
activity (entry 6). Interestingly, difference in these phosphotransferase inhibitory profiles between CPPB
and I-CPPB correlate with their antimycobacterial activity: CPPB possessing MraY/WecA inhibitory
activity killed Myocobacterium tuberculosis H37Rv, Mycobacterium avium 2285, Mycobacterium
smegmatis (ATCC607) with the MIC values 6.25-12.5 g/mL. In contrast, I-CPPB, which does not have
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MraY inhibitory activity, did not show growth inhibitory activity against these Mycobacterium spp. at 50
g/mL.

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Insert Scheme 1 and Scheme 2
Insert Table 1
Cytotoxicity of new capuramycin analogues, CPPB (5) and I-CPPB (6). In the capuramycin analogue
series, the degree of MraY inhibitory activity correlates with their antimycobaterial activity.7-9,13
Antimycobacterial capuramycin analogues display low in vitro cytotoxicity against mammalian cells, and
have been recognized as safe drug leads that have acceptable tolerability in animal models.2,3 The toxicity
of tunicamycin (11, Figure 1) has been studied extensively in vitro: tunicamycin inhibits growth in many
cancer cell lines without selectivity, and has a narrow therapeutic window demonstrated in in vivo studies
using mice.18,29 The toxicity of tunicamycin is believed to be attributable to its ability to inhibit DPAGT1
enzyme function.10,30 However, in our studies, tunicamycin’s toxicity could not be explained solely by its
inhibition of DPAGT1. Our DPAGT1 inhibitor, APPB (12, Figure 1) inhibits DPAGT1 with greater than
30-times the inhibitory activity of tunicamycin, and inhibits growth of selected solid cancer cell lines at low
10
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M concentrations with selective cytotoxicity (IC50 normal cells / cancer cells) of >35. The LD50 value of
APPB is >20 mg/kg (mouse), whereas it is 2.0 mg/kg (mouse) for tunicamycin.10,18 Capuramycin-based
DPAGT1 inhibitors, CPPB (5) and I-CPPB (6), identified in this program inhibited DPAGT1 enzyme with
the IC50 values of 0.2 and 0.6 M, respectively. Unlike the MraY-antimycobacterial activity relationship
observed for CAP analogues, the DPAGT1 inhibitors, CPPB and I-CPPB, did not show antiproliferative
activity against L1210 (a leukemia cell), HPNE (a normal pancreatic ductal cell), and Vero (a normal kidney
cell) at 50 M. They showed various levels of growth inhibitory activity against several solid cancer cell
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lines such as KB (HeLa, a cervix carcinoma), SiHa (a cervical squamous cell carcinoma), HCT-116 (a
colorectal adenocarcinoma), DLP-1 (a colorectal adenocarcinoma), Capan-1 (a pancreatic ductal
adenocarcinoma), PANC-1 (a pancreatic ductal carcinoma), AsPC-1 (a pancreatic adenocarcinoma), PD002
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(a patient-derived pancreatic adenocarcinoma) in MTT assays (IC50 15-45 M, Table 2). A lower DPAGT1
inhibitor, tunicamycin (11), showed growth inhibition of all cell lines in Table 2 with the IC50 values of
0.78-7.5 M concentrations (entry 5 in Table 2). Cellular behavior and morphological changes of a patient-
derived metastatic pancreatic adenocarcinoma, PD002 treated with CPPB were monitored over time via
®
IncuCyte live cell analysis imaging system (Figure 4A). Interestingly, 10-13% of phase area confluent of
PD002 culture (time 0h) remained the same after 72h for the CPPB-treated cells (50 M), whereas, ca. 70%
of confluence was reached for the control PD002 culture (PBS) (Figure 4B vs.4C). Although morphological
changes were subtle over time (0-72h), cell viability assessed by the MTT reduction assay revealed that
PD002 cells treated with CPPB (50 M) was significantly decreased (Table 2). Exposure of CPPB (0.2-20
M) inhibited cell proliferation of PD002: ca. 20% of cell proliferation was inhibited at time 72h. These
results may indicate that DPAGT1 inhibitors may have cytostatic effect again certain cancerous tumors that
require DPAGT1 overexpression for their growth.
Insert Table 2
Insert Figure 4
Cell migratory inhibition by CPPB (5). DPAGT1 catalyzes the first step in N-glycan biosynthesis of
mammalian cells (Figure 2). Aberrant N-glycosylation is common in many solid cancers and important for
the epithelial to mesenchymal transition program (EMT, a mechanism of metastases).10 We found high
levels of DPAGT1 protein expression in a series of pancreatic cancers (e.g., PANC-1, Capan-1, and AsPC-
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). Dysregulation of DPAGT1 enzyme leads to disturbances in cell-cell adhesions and may increase
epithelial to mesenchymal transition (EMT): these processes increase migratory and invasive capabilities of
malignant neoplasms that are the initiation of metastasis in cancer progression, especially pancreatic
cancer.31,32 Interestingly, there is significant crosstalk between DPAGT1 and the Wnt/-catenin and Snail
pathway where DPAGT1 overexpression leads to 1) accumulation of -catenin in the cytoplasm and then
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translocation into the nucleus, and 2) reduction of the Snail expression levels, preventing epithelial-
mesenchymal transition by suppressing the E-cadherin expression (a cell-cell adhesion glycoprotein).10,31,32
As such, aberrations in these pathways occur in numerous cancers, thus, discovery of small molecules
directed towards inhibition of the Wnt and Snail pathways represents an important area of anticancer
therapeutics.33-35 In order to obtain insights into anti-metastatic ability of DPAGT1 inhibitors, we explored
the degree of cell migration in several commercially available cell lines (Capan-1, PANC-1, and AsPC-1),
a patient-derived pancreatic ductal adenocarcinoma cell line (PD002), a cervical carcinoma (SiHa), and a
colorectal adenocarcinoma (HCT-116) to determine the effects on cellular motility. After 24h of three
treatment doses (0.05, 0.1, and 0.2 M) with CPPB (5), inhibition of migration (closing the gap) was
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measured in a scratch assay (wound healing assay). Only the images for PD002 are shown in Figure 5 and
all other images obtained through these experiments are illustrated in Supporting Information (SI). The
pancreatic cancer cell lines treated with CPPB migrated far less than the PBS treated (control) cells (Figure
5
A-D). In these assays, the wound-healing rate of the untreated PD002 cells was 63% in 24h. In sharp
contrast, CPPB treated cells inhibited the wound-healing effectively at its IC50 level against DPAGT1 (0.2
M): the wound-healing rate was approximately 20% (Figure 5B). We thoroughly evaluated migration

inhibition ability of CPPB compared to gemcitabine, one of the main chemotherapy drugs used to treat
pancreatic cancer, and tunicamycin using PD002 cells. Gemcitabine shows a wound-healing rate of 43% at
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.2 M, and tunicamycin shows 35% at 0.2 M (SI). Thus, it was concluded that CPPB is more effective in
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inhibiting cancer cell migration than gemcitabine and tunicamycin. These trends were further confirmed by
an endpoint migration assays via Boyden chambers for PD002.37,38 In these assays, the cell migrations of
PD002 treated with CPPB (0.1 M) were inhibited on a higher level compared to those with tunicamycin
and gemcitabine at the same concentration (0.1 M) (Figure 6).
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Insert Figure 5
Insert Figure 6
Inhibition of a zinc-finger transcription factor, Snail1 (Snail) in the selected cancer cell lines by
CPPB. Snail protein is one of the most important transcription factor that induces epithelial to mesenchymal
transition (EMT), which converts epithelial cells into migratory mesenchymal cells that are more efficient
at metastasizing.39 EMT induced by overexpression of Snail produces cancer stem-like properties in a
number of solid organ cancers. Aberrant expression of Snail leads to loss of expression of E-cadherin.40
Thus, suppression of Snail expression or inhibition of Snail functions represents a potent targeted
therapeutic strategy for many cancers.41,42
Immunofluorescence assays using an anti-Snail antibody revealed that the fluorescence intensity of Snail
was strong in a series of pancreatic cancer cells (PANC-1, AsPC-1, Capan-1, and PD002), and the
expression of Snail was decreased by the treatment with CPPB in a concentration dependent manner. Among
pancreatic cancer cell lines, only the data for PD002 and PANC-1 are shown in Figure 7A and 7B (see SI
for AsPC-1 and Capan-1). The Snail expression level of a non-metastatic pancreatic cancer, PANC-1, was
much lower than metastatic pancreatic cancers (e.g., PD002 and Capan-1). A few other types of cancer cells
such as a colorectal cancer (HCT-116) and a cervical cancer (SiHa) were examined by similarly designed
immunofluorescence assays or Western blot assays (Figures 7C and 7D). The Snail expression in SiHa was
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inhibited by treatment of CPPB in a concentration dependent manner; at the IC50 concentration (0.2 M
against DPAGT1), CPPB effectively inhibited the Snail expression (Figure 7D). In contrast, the Snail
expression level in HCT-116 was not noticeably changed by the treatment of CPPB between 0.05 and 2.0
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M concentrations. Interestingly, cell migration of HCT-116 was not inhibited by CPPB demonstrated in
the wound healing (scratch) assays (Figure 5F). At the concentrations tested in the scratch assays (0.05-2.0
M), the E-Cadherin expression levels of PD002, PANC-1, and HCT-116 were not changed significantly
Figure 8). To support the above discussion based on the immunefluorescent staining (Figure 7 and 8), the

(
relative expression levels of Snail and E-cadherin in PD002, PANC-1, and HCT-116 treated with CPPB
were measured by Western blot analyses (Figure 9). The relative expression levels were obtained by using
Image Studio™ Lite quantification software, and these quantified data were summarized in SI (see Figure
S5). PD002 and PANC-1 treated with CPPB (0 to 2.0 M) lead to a dose dependent decrease in Snail. The
E-Cadherin expression level of a metastatic pancreatic cancer, PD002 was increased in a CPPB
concentration depended manner (0 to 2.0 M), whereas, a non-metastatic pancreatic cancer, PANC-1
exhibited the same expression level of E-Cadherin at 0-2.0 M concentrations of CPPB (Figure 9A and 9B).
A low DPAGT1 expressed-colon cancer, HCT-116 did not display noticeable difference in the expression
levels of Snail and E-Cadherin at 0-2.0 M CPPB concentrations (Figure 9C).
Insert Figure 7
Insert Figure 8
Insert Figure 9
Inhibition of DPAGT1 by CPPB. CPPB decreased the DPAGT1 expression level in all pancreatic cancer
cell lines examined in Figure 5: the DPAGT1 expression was apparently inhibited by the IC50 concentration
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of CPPB (0.2 M) (only the data for PD002 and PANC-1 shown Figure 10 and see Figure S4 in SI) for
AsPC-1 and Capan-1). An important observation is that the DPAGT1 expression of the pancreatic cancer
cell lines could not completely be inhibited at a high concentration of CPPB (2-20 M) (Figures 9 and 10).
In MTT assays, we realized that all pancreatic cancers tested remained viable at 20 M concentration of
CPPB (Table 2). We confirmed that the DPAGT1 expression level in a colorectal adenocarcinoma, HCT-
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16 is significantly lower than that in the pancreatic cancer cell lines (PD002 and PANC-1); DPAGT1 was
not detectable in Western blot assays for the lysate obtained by a standard protocol (50 g/30 L of total
protein sample). A 10 times concentrated HCT-116 cell lysate (prepared by ultracentrifugation, 130,000xg
for 1h) enabled us to detect DPAGT1 in Western blotting. By treatment of HCT-116 with CPPB at 0.1-20
M concentration, the DPAGT1 expression levels of HCT-116 remained higher fluorescence intensity in
immunefluorescent assay (Figure 10C) and 20-90% in Western blot assays (Figure 9C). These data imply
that the inhibitory effect of CPPB on cell migration varies depending on degree of inhibition of the DPAGT1
expression: immunefluorescent assays at 0.2-2.0 M concentrations of CPPB, the degree of DPAGT1
expression was decreased by the following order: PANC-1 > PD002 >> HCT-116. Migration inhibition
observed in the scratched assays (Figure 5) is well-correlated with the degree of the DPAGT1 expression
inhibition. Although CPPB decreased the protein expression of DPAGT1 without significantly decreasing
its gene (DPAGT1) expression, tunicamycin decreased both the gene expression of DPAGT1 and DPAGT1
protein expression (Figure 11). These down-regulation in DPAGT1 gene expression by tunicamycin may be
attributable to its high cytotoxicity against mammalian cells without selectivity.
Insert Figure 10 and Figure 11
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Synergistic effect of CPPB with paclitaxel. The FOLFIRINOX (a combination of folinic acid, 5-
fluorouracil, irinotecan, and oxaliplatin) and nab-paclitaxel (albumin-bound paclitaxel)-gemcitabine
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regimens have been adopted into clinical practice for patients with metastatic pancreatic cancers. Median
progression-free survival was reported in one study of patients with metastatic pancreatic cancer to be 6.4
months in the FOLFIRINOX group and 3.3 months in the gemcitabine group.43,44 Over the past years, the
clinical data have not supported that FOLFIRINOX is associated with any better (or worse) survival rates
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compared to the nab-paclitaxel-gemcitabine regimen as there have been no head-to-head trials. However,
the inclusion of paclitaxel and its derivatives in combination regimens remains an important therapeutic
strategy in pancreatic cancer chemotherapy since nab-paclitaxel-gemcitabine is associated with less adverse
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effects (toxicity in patients) than FOLFIRINOX. In this regard, we were very interested in synergistic or
additive effects of DPAGT1 inhibitor in combination with paclitaxel. The synergistic or antagonistic
activities of CPPB were assessed in vitro via checkerboard technique.47,48 In these experiments, CPPB
displayed strong synergistic effects with paclitaxel in a wide range of concentrations against PD002. Table
3
summarizes the results of FIC index analyses for selected combinations of CPPB (IC50 35.0 M) plus
paclitaxel (IC50 1.25 M) that showed synergistic combination (FIC<0.5). The FIC index below 0.50 was
observed for 20 combinations of two molecules out of 96 different concentrations (see Figure S6 in SI). The
IC50 value of paclitaxel against PD002 was lowered (0.024-0.61 M) in combination with CPPB (0.1-2.0
M).
Insert Table 3
Interaction of CPPB with DPAGT1 (Modeling Studies). DPAGT1 encompasses ten transmembrane
segments, three loops on the ER (endoplasmic reticulum) side, and five loops on the cytoplasmic side. Four
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loops (CL-1, CL-5, CL-7, and CL-9) on the cytoplasmic side form the UDP-GlcNAc-binding domain.25,26
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The hydrophobic tunnel created by the transmembrane segments (TM-4, TM-5 and CL-9) within the lipid
bilayer is predicted to interact with dolychol-phosphate (Dol-P). The weak DPAGT1 inhibitors, O-methyl
capuramycin (OM-CAP) and iso-capuramycin (I-CAP) (Table 1), yielded low docking scores (Glide
Scores) using Schrödinger’s Glide program: the score for OM-CAP was -12.4 and for I-CAP was -10.9.
These scores predicted that OM-CAP and I-CAP possess significantly lower affinity for DPAGT1 than
CPPB, which showed a docking score of -16.6 (see Table S1 in SI).49-51 The docked CPPB-DPAGT1
structure illustrated in Figure 12 shows several key interactions. The C2’-OH acts as a donor in a hydrogen
bond to the Glu56 carboxylate. This interaction is likely be lost when the C2’-OH is methylated. Pi-stacking
interactions are observed between 1) Phe249 and the uracil ring, and 2) Trp122 and the
trifluoromethoxybenzene in the hydrophobic moiety. Asn185 and Ash252 (protonated Asp) form hydrogen-
bond(s) to the primary amide and the dihydropyran-hydroxy (C3”-OH) group, respectively. Additional
hydrogen-bonds between the uridine ureido group and the backbone amides (Gln44 and Leu46) strengthen
the ligand interaction. In this program, capuramycin (CAP), a strong MraY inhibitor with no DPAGT1
inhibitory activity, was successfully engineered to be a relatively strong DPAGT1 inhibitor by introducing
a hydrophobic functional group, (((trifluoromethoxy)phenoxy) piperidin-1-yl)phenyl)methylamine at the
C6’’-position. This hydrophobic group allows the preferable conformation of the uridine-
enopyranosiduronic moiety in the DPAGT1 biding domain, resulting in increased interactions with Asn185,
Ash252, Glu56, and Phe249.
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Insert Figure 12
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A semi-synthesis of CPPB from A500359F. Pharmacological studies of CPPB (5) and its related
analogues using appropriate animal models will be a focus of our future research efforts. Previously, a
natural product A-5003659E was used to develop novel capuramycin analogues with strong MraY
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inhibitory activity (Hotoda et. al. 2003) (Figure 3). Its free-carboxylic acid analogue, A-500359F was also
isolated from the capuramycin-producing strain, Streptomyces griseus Sank 60196. Saponification of A-
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003659E to A-500359F was established by the Sankyo group. Although, the currently available synthetic
schemes for capuramycin analogues (e.g., Scheme 3) include a relatively short number of chemical
steps,22,23,53 a semi-synthetic approach is more feasible to deliver a large quantities of CPPB for
pharmacological studies. To establish semi-synthesis of CPPB, A-500359F was first synthesized from the
CAP-synthetic intermediate 32 (Scheme 3A) in a single step. Amide-forming reaction of synthetic A-
5
00359F with (((trifluoromethoxy)phenoxy) piperidin-1-yl)phenyl)methylamine (34) was performed under
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an optimized condition using EDCI, glyceroacetonide-Oxyma, and NMM in DMF. All coupling reagents
could be removed by partitions between CHCl
through DOWEX 50Wx4 column (MeOH : NH
further purified by C18-reverse HPLC (MeOH : H
3
and water and evaporation. The crude product was passed
4
OH = 4 : 1) to provide CPPB with >95% purity, which was
2
O = 65 : 35) to yield pure CPPB (Scheme 4).
Insert Scheme 4
CONCLUSION
This paper describes the identification of a new DPAGT1 inhibitor of capuramycin analogue,
capuramycin phenoxypiperidinylbenzylamide (CPPB, 5) and its isostere I-CPPB (6). Previously,
tunicamycin is the only DPAGT1 inhibitor that has been widely applied to the studies associated with
protein misfolding in vitro.55,56 Tunicamycin displays cytotoxicity against cancer and healthy cells with low
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selectivity ratio. One of cytotoxicity mechanisms of tunicamycin is believed to be its ability to inhibit
DPAGT1 enzyme functions. The DPAGT1 expression levels vary depending on the cell types; renal cancers
and lymphomas express low-levels of DPAGT1, whereas, a majority of solid cancers express high-levels.57
Thus, the observed antiproliferative activity of tunicamycin against all types of cancer cells are difficult to
understand solely by its DPAGT1 inhibitory activity. In our studies, CPPB showed ~7.5 times stronger
DPAGT1 inhibitory activity than tunicamycin. However, unlike tunicamycin, CPPB did not inhibit growth
of cancer cell lines at the IC50 values observed for tunicamycin (0.45 -7.5 M). CPPB is a cell-permeable
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molecule which was demonstrated by IncuCyte live cell analyses and immunofluorescence assays. In this
article, we have studied effectiveness of CPPB on the expression levels of Snail, E-cadherin, and DPAGT1
primary in pancreatic cancers (Figures 7-10). CPPB decreased the Snail expression in commercially
available pancreatic cancer cell lines (PANC-1, AsPC-1, and Capan-1) and a patient-derived pancreatic
ductal adenocarcinoma cell line (PD002) in a dose dependent manner. On the other hand, the E-cadherin
expression level was increased in PD002 (a metastatic pancreatic cancer cell) or was not noticeably changed
in PANC-1 at between 0.05-0.2 M of CPPB. These biochemical data may support that a selective DPAGT1
inhibitor, CPPB is effective in inhibiting metastasis spread of the pancreatic cancer cells observed in scratch
and transwell assays (Figures 5 and 6). Other than pancreatic cancers, a lower DPAGT1 expression cell, a
colorectal adenocarcinoma (HCT-116) and a higher DPAGT1 expression cell, a cervical carcinoma (SiHa)
were examined. CPPB did not inhibit migration of HCT-116, but strongly inhibited migration of SiHa in
scratch assays at 0.2 M (IC50 concentration against DPAGT1). These observations were supported by the
biochemical analyses of the Snail and E-cadherin expression levels. Snail plays an important role in cancer
progression. The accumulated evidences on Snail indicate that over-expression of Snail promotes drug
resistance, tumor recurrence and metastasis.40 Although, only limited data have been generated in this
article, CPPB’s Snail inhibitory activity observed in pancreatic and a several high DPAGT1-expressed cell
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lines suggests that selective DPAGT1 inhibitors have the potential to develop into less toxic anticancer
therapeutics than anticancer drugs that are cytotoxic to all dividing cells in the body. CPPB is not cytotoxic
against a series of cancer and healthy cell lines at 10 M or higher concentrations. However, it showed a
cytostatic activity against pancreatic cancers and a strong synergistic effect with paclitaxel; cytotoxic
activity of paclitaxel was improved over 250-times against PD002 in combination with CPPB (0.2-2.0 M).
Docking studies of CPPB with the available DPAGT1 crystal structures provided insight into unique
interactions, and thus, structure-based molecule design may be a fruitful approach to improve CPPB’s
DPAGT1 affinity. A collaboration with Daiichi-Sankyo is essential to efficiently produce CPPB for in vivo
studies using large-animal models such as dogs, pigs, and monkeys. We have demonstrated a semi-synthesis
of CPPB from a capuramycin biosynthetic intermediate, A-500359F, that will secure a production of large
amount of CPPB (Scheme 4). Our total synthetic scheme is amenable to produce gram-quantity of CPPB
(Scheme 3). Extensive synergistic, toxicity, and pharmacokinetic studies of CPPB will be performed using
preclinical animal models, and these data including detailed evaluation on in vivo efficacy against pancreatic
and cervical cancers will be reported elsewhere.
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EXPERIMENTAL SECTION
Chemistry. General Information. All chemicals were purchased from commercial sources and used
without further purification unless otherwise noted. THF, CH
2
Cl , and DMF were purified via Innovative
2
Technology's Pure-Solve System. All reactions were performed under an Argon atmosphere. All stirring
was performed with an internal magnetic stirrer. Reactions were monitored by TLC using 0.25 mm coated
commercial silica gel plates (EMD, Silica Gel 60F254). TLC spots were visualized by UV light at 254 nm,
or developed with ceric ammonium molybdate or anisaldehyde or copper sulfate or ninhydrin solutions by
heating on a hot plate. Reactions were also monitored by using SHIMADZU LCMS-2020 with solvents: A:
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.1% formic acid in water, B: acetonitrile. Flash chromatography was performed with SiliCycle silica gel
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
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3
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4
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4
5
5
5
5
5
5
5
5
5
5
6
1
(
Purasil 60 Å, 230-400 Mesh). Proton magnetic resonance ( H-NMR) spectral data were recorded on 400,
1
3
and 500 MHz instruments. Carbon magnetic resonance ( C-NMR) spectral data were recorded on 100 and
0
1
2
3
4
5
6
7
8
9
0
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6
7
8
9
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9
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4
5
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7
8
9
0
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2
3
4
5
6
7
8
9
0
1
25 MHz instruments. For all NMR spectra, chemical shifts (δH, δC) were quoted in parts per million (ppm),
1
13
and J values were quoted in Hz. H and C NMR spectra were calibrated with residual undeuterated solvent
CDCl : δH = 7.26 ppm, δC = 77.16 ppm; CD CN: δH = 1.94 ppm, δC = 1.32ppm; CD OD: δH =3.31 ppm,
δC =49.00 ppm; DMSO-d : δH = 2.50 ppm, δC = 39.52 ppm; D O: δH = 4.79 ppm) as an internal reference.
(
3
3
3
6
2
The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, dd = double
doublets, t = triplet, q = quartet, quin = quintet, hept = heptet, m = multiplet, br = broad. Infrared (IR) spectra
were recorded on a Perkin-Elmer FT1600 spectrometer. HPLC analyses were performed with a Shimadzu
LC-20AD HPLC system. HR-MS data were obtained from a Waters Synapt G2-Si (ion mobility mass
spectrometer with nanoelectrospray ionization). All assayed compounds were purified by reverse HPLC to
be ≥95% purity.
(
2S,3S,4S,5R,6R)-2-((1S)-((2R,5R)-3-Acetoxy-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)(cyano)methoxy)-6-((2-
chloroacetoxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15). To a stirred suspension of 13
(
0.17 g, 0.38 mmol), 14 (0.37 g, 0.75 mmol), MS3Å (0.50 g) and SrCO
mL) were added AgBF (0.037 g, 0.19 mmol) and NIS (0.25 g, 1.13 mmol) at 0 °C. After being stirred for
9h, the reaction mixture was added Et N (1.0 mL) and passed through a silica gel pad (hexanes/EtOAc =
3
(0.28 g, 1.88 mmol) in CH
2
Cl (9.4
2
4
1
3
1/4). The filtrate was concentrated in vacuo. The crude mixture was purified by silica gel column
1
chromatography (hexanes/EtOAc = 2/1 to 1/2) to afford 15 (0.24 g, 78%): H NMR (400 MHz, Chloroform-
d) δ 7.40 – 7.27 (m, 6H), 6.07 (d, J = 4.7 Hz, 1H), 6.00 (d, J = 8.2 Hz, 1H), 5.51 (d, J = 9.8 Hz, 1H), 5.47
(d, J = 9.6 Hz, 1H), 5.37 (dd, J = 3.4, 2.0 Hz, 1H), 5.28 (d, J = 10.2 Hz, 1H), 5.22 – 5.15 (m, 2H), 4.84 (d,
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1
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2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
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3
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J = 3.2 Hz, 1H), 4.71 (s, 2H), 4.41 (dd, J = 5.5, 3.2 Hz, 1H), 4.39 – 4.35 (m, 1H), 4.33 (d, J = 6.0 Hz, 1H),
.27 (d, J = 2.3 Hz, 1H), 4.14 (d, J = 1.7 Hz, 2H), 4.03 – 4.01 (m, 1H), 3.93 (ddd, J = 8.9, 5.9, 2.4 Hz, 1H),
4
1
3
3.47 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H); C NMR (101 MHz, CDCl
3
) δ 170.20,
69.84, 169.50, 166.92, 162.19, 150.97, 137.75, 137.46, 128.32 (2C), 127.72, 127.69 (2C), 113.96, 103.58,
6.29, 88.68, 80.93, 80.09, 72.30, 70.42, 69.60, 68.46, 67.94, 65.16, 64.35, 63.58, 59.26, 40.61, 31.58,
0.70, 20.66, 20.62, 20.57; HRMS (ESI+) m/z calcd for C35 17 [M + H] 810.2125, found: 810.2151.
2R,3S,4S,5R,6R)-2-((1R)-1-((2S,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-
triyl triacetate (16). To a stirred solution of 15 (0.24 g, 0.29 mmol) in a 9:1 mixture of EtOH and H O (2.9
mL) were added HgCl (0.16 g, 0.59 mmol) and acetaldoxime (0.18 mL, 2.9 mmol). After being stirred for
3h at r.t., the reaction mixture was concentrated in vacuo. The residue was quenched with aq. NaHCO
and extracted with CHCl . The combined organic extracts were dried over Na SO and concentrated in
vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 1/2 to
CHCl /MeOH = 96/4) to afford the amide (0.21 g, 87%). To a solution of the amide (0.21 g, 0.26 mmol) in
a 1:1 mixture of THF and MeOH (2.6 mL) was added thiourea (0.059 g, 0.77 mmol). After being stirred for
1h at 50 °C, the reaction mixture was concentrated in vacuo. The residue was diluted with H O and
extracted with CHCl . The combined organic extracts were dried over Na SO and concentrated in vacuo.
The crude product was purified by silica gel column chromatography (CHCl /MeOH = 98/2 to 97/3 to 96/4)
0
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1
9
2
H
41ClN O
3
(
2
2
1
3
,
3
2
4
3
1
2
3
2
4
3
to afford the primary alcohol (0.15 g, 75%). To a stirred solution of the primary alcohol (0.15 g, 0.19 mmol)
i
and AcOH (0.040 mL) in a 1:1 mixture of THF and PrOH (2.0 mL) was added 10% Pd/C (0.12 g) under
N
2
. H
2
gas was introduced and the reaction mixture was stirred under H atmosphere for 3h, the solution
2
was filtered through Celite and concentrated in vacuo. The crude product was purified by silica gel column
1
chromatography (CHCl
3
/MeOH = 97/3 to 92/8) to afford 16 (0.10 g, 81%): H NMR (400 MHz,
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Chloroform-d) δ 9.08 (brs, 1H), 7.60 (d, J = 8.2 Hz, 1H), 6.87 (brs, 1H), 6.12 (brs, 1H), 6.00 (d, J = 8.1 Hz,
H), 5.87 (d, J = 3.6 Hz, 1H), 5.55 – 5.52 (m, 1H), 5.26 – 5.22 (m, 2H), 5.20 (t, J = 6.0 Hz, 1H), 5.00 (d, J
= 1.7 Hz, 1H), 4.53 (dd, J = 6.5, 2.3 Hz, 1H), 4.46 (d, J = 2.3 Hz, 1H), 4.06 (dd, J = 5.5, 3.7 Hz, 1H), 3.76
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
–
3.71 (m, 1H), 3.69 – 3.57 (m, 2H), 3.44 (s, 3H), 2.17 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H); C
NMR (101 MHz, CDCl ) δ 171.42, 170.46, 170.46, 170.40, 163.10, 150.03, 139.50, 103.61, 97.15, 96.95,
8.50, 81.26, 80.96, 75.33, 72.91, 69.67, 68.89, 65.54, 61.27, 59.05, 50.86, 20.82, 20.75, 20.67, 20.59;
HRMS (ESI+) m/z calcd for C25 16 [M + H] 632.1939, found: 632.1963.
3
8
H
34
3
N O
(
2S,3S,4S)-3,4-Diacetoxy-2-((1R)-1-((2S,5R)-3-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-4-methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-3,4-dihydro-2H-pyran-6-carboxylic acid
18). To a stirred solution of 16 (0.10 g, 0.16 mmol) and DMSO (0.11 mL, 1.57 mmol) in a 5:1 mixture of
CH Cl and Et N (0.80 mL) was added SO •pyridine (0.25 g, 1.57 mmol). After being stirred for 3h at r.t.,
the reaction mixture was added H O (0.16 mL) and passed through a silica gel pad (CHCl /MeOH = 92/8)
(
2
2
3
3
2
3
t
to provide the crude 17. To a stirred solution of the crude mixture in BuOH (1.0 mL) and 2-methyl-2-butene
(0.5 mL) was added a solution of NaClO
2
(0.071 g, 0.78 mmol) and NaH
2
PO
4
•2H
2
O (0.12 g, 0.78 mmol) in
O and extracted with
H
2
O (1.0 mL). After being stirred for 4h at r.t., the reaction was quenched with H
2
CHCl
3
/MeOH (9/1). The combined organic extracts were dried over Na
2
SO
4
and concentrated in vacuo.
/MeOH = 9/1) to afford 18
The crude product was purified by silica gel column chromatography (CHCl
3
1
(
0.078 g, 85%): H NMR (400 MHz, Methanol-d
4
) δ 7.77 (d, J = 8.1 Hz, 1H), 5.94 (d, J = 11.7 Hz, 1H),
5
5
3
.94 (s, 1H), 5.78 (t, J = 2.1 Hz, 1H), 5.66 (dd, J = 4.5, 2.5 Hz, 1H), 5.56 (ddd, J = 4.7, 3.2, 1.6 Hz, 1H),
.32 (d, J = 3.3 Hz, 1H), 4.99 (t, J = 5.2 Hz, 1H), 4.85 (d, J = 2.1 Hz, 1H), 4.60 (dd, J = 5.2, 2.0 Hz, 1H),
13
.95 (t, J = 5.0 Hz, 1H), 3.39 (s, 3H), 2.13 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H); C NMR (101 MHz, MeOD)
δ 172.79, 172.06, 171.60, 167.79, 166.01, 152.05, 148.16, 140.97, 104.23, 103.98, 97.91, 88.13, 83.38,
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2.91, 76.56, 71.95, 65.17, 65.06, 59.41, 20.75, 20.63, 20.57; HRMS (ESI+) m/z calcd for C23
H
28
N O15 [M
3
+
H] 586.1520, found: 586.1549.
(2S,3S,4S)-2-((1R)-1-((2S,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-(((S)-2-oxoazepan-3-yl)carbamoyl)-3,4-
0
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8
9
0
1
2
3
4
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dihydro-2H-pyran-3,4-diyl diacetate (19). To a stirred solution of 18 (31 mg, 0.053 mmol), 2-(S)-
aminocaprolactam (26 mg, 0.16 mmol), HOBt (21 mg, 0.16 mmol) and NMM (58 L, 0.53 mmol) in DMF
(0.26 mL) was added EDCI (51 mg, 0.26 mmol). After being stirred for 6h at r.t., the reaction was quenched
with aq. NaHCO , and extracted with CHCl /MeOH (9/1). The combined organic extracts were dried over
3
3
Na
2
SO
4
and concentrated in vacuo. The crude product was purified by silica gel column chromatography
1
(CHCl
3
/MeOH = 95/5) to afford 19 (30 mg, 82%): H NMR (400 MHz, Methanol-d
4
) δ 7.68 (d, J = 8.2 Hz,
1
1
H), 6.02 (t, J = 2.1 Hz, 1H), 5.91 (d, J = 4.2 Hz, 1H), 5.89 (d, J = 6.3 Hz, 1H), 5.73 (dd, J = 4.4, 2.5 Hz,
H), 5.68 – 5.65 (m, 1H), 5.51 (d, J = 3.0 Hz, 1H), 5.01 (d, J = 4.6 Hz, 1H), 4.76 (d, J = 2.0 Hz, 1H), 4.62
(d, J = 12.5 Hz, 1H), 4.60 – 4.57 (m, 1H), 3.98 (d, J = 6.1 Hz, 1H), 3.35 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H),
13
2
.05 (s, 3H), 2.03 – 1.98 (m, 2H), 1.91 – 1.83 (m, 2H), 1.65 – 1.56 (m, 2H), 1.44 – 1.37 (m, 2H); C NMR
(101 MHz, MeOD) δ 176.12, 172.32, 171.66, 171.29, 171.21, 165.96, 160.96, 151.90, 144.61, 141.42,
106.25, 103.54, 98.81, 88.77, 83.41, 79.28, 77.39, 74.79, 64.91, 64.49, 59.26, 57.36, 53.25, 42.36, 32.19,
2
6
9.75, 28.94, 20.54, 20.41, 20.32; HRMS (ESI+) m/z calcd for C29
H
38
5
N O15 [M + H] 696.2364, found:
96.2391.
(
2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-
methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-((S)-2-oxoazepan-3-yl)-3,4-dihydro-
H-pyran-6-carboxamide (3). A solution of 19 (30 mg, 0.043 mmol) in a 5:1 mixture of MeOH and Et
0.60 mL) was stirred for 5h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-
2
3
N
(
phase HPLC [column: Luna® (C18, 10 m, 100 Å, 250 x 10 mm), solvents: 15:85 MeOH:H O, flow rate:
2
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2
3
1
3
.0 mL/min, UV: 254 nm, retention time: 20 min] to afford I-CAP (3, 24 mg, 98%): H NMR (400 MHz,
Methanol-d ) δ 7.92 (d, J = 8.1 Hz, 1H), 6.02 (d, J = 3.8 Hz, 1H), 5.88 (d, J = 5.1 Hz, 1H), 5.74 (d, J = 8.1
Hz, 1H), 5.23 (d, J = 5.6 Hz, 1H), 4.67 (d, J = 2.0 Hz, 1H), 4.59 – 4.54 (m, 2H), 4.38 (t, J = 4.2 Hz, 1H),
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
1
1
6
5
.29 (t, J = 5.1 Hz, 1H), 3.98 (t, J = 5.0 Hz, 1H), 3.84 (t, J = 4.6 Hz, 1H), 3.43 (s, 3H), 2.06 – 1.99 (m, 2H),
13
.89 – 1.81 (m, 2H), 1.62 – 1.45 (m, 2H), 1.44 – 1.33 (m, 2H); C NMR (101 MHz, MeOD) δ 176.27,
73.46, 166.15, 161.85, 152.32, 144.23, 141.91, 109.37, 102.82, 101.22, 90.27, 83.49, 81.02, 78.93, 74.54,
8.51, 63.53, 58.67, 53.35, 42.48, 32.36, 29.91, 29.06; HRMS (ESI+) m/z calcd for C23
H
32
5
N O12 [M + H]
70.2048, found: 570.2071.
(
2S,3S,4S,5R,6R)-2-((S)-((3aR,4R,6R,6aR)-6-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)(cyano)methoxy)-6-
(
(2-chloroacetoxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (21). To a stirred suspension of 20
0.24 g, 0.56 mmol), 14 (0.55 g, 1.12 mmol), MS3Å (0.72 g), and SrCO (0.41 g, 2.80 mmol) in CH Cl
(0.055 g, 0.28 mmol) and NIS (0.25 g, 1.12 mmol) at 0 °C. After being stirred
for 12h, the reaction mixture was added Et N (1.0 mL), and passed through a silica gel pad (hexanes/EtOAc
1/4). The filtrate was concentrated in vacuo. The crude mixture was purified by silica gel column
(
3
2
2
(14.0 mL) were added AgBF
4
3
=
1
chromatography (hexanes/EtOAc = 6/4 - 4/6) to afford 21 (0.39 g, 88%): H NMR (400 MHz, Chloroform-
d) δ 7.37 – 7.28 (m, 5H), 7.20 (d, J = 8.1 Hz, 1H), 5.82 (d, J = 8.1 Hz, 1H), 5.61 (d, J = 1.6 Hz, 1H), 5.50
(
d, J = 9.8 Hz, 1H), 5.42 (d, J = 9.8 Hz, 1H), 5.35 – 5.27 (m, 1H), 5.23 (dd, J = 10.0, 3.3 Hz, 1H), 5.07 (d,
J = 1.9 Hz, 1H), 5.00 (d, J = 3.5 Hz, 1H), 4.98 (dd, J = 6.4, 1.6 Hz, 1H), 4.84 (d, J = 7.1 Hz, 1H), 4.69 (s,
2H), 4.43 (dd, J = 7.1, 3.6 Hz, 1H), 4.20 (dd, J = 12.2, 4.3 Hz, 1H), 4.17 – 4.07 (m, 2H), 4.05 (d, J = 1.6
13
Hz, 2H), 3.97 – 3.92 (m, 1H), 2.16 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.59 (s, 3H), 1.39 (s, 3H); C NMR
(
101 MHz, CDCl ) δ 169.80, 169.57, 169.47, 166.80, 162.08, 150.84, 140.89, 137.67, 128.33 (2C), 127.91,
3
127.77, 127.63 (2C), 115.23, 114.72, 102.84, 96.56, 96.30, 86.55, 84.15, 80.95, 72.42, 70.41, 69.41, 68.39
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5
5
6
(
2C), 66.04, 65.35, 62.90, 40.52, 27.03, 25.20, 20.73, 20.64, 20.59; HRMS (ESI+) m/z calcd for
16 [M + H] 794.2175, found: 794.2198.
(2R,3S,4S,5R,6R)-2-((R)-2-Amino-1-((3aR,4S,6R,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-oxoethoxy)-6-(hydroxymethyl)tetrahydro-
H-pyran-3,4,5-triyl triacetate (22). To a stirred solution of 21 (0.39 g, 0.49 mmol) in a 9:1 mixture of
EtOH and H O (4.9 mL) were added HgCl (0.27 g, 0.98 mmol) and acetaldoxime (0.30 mL, 4.9 mmol).
After being stirred for 12h at r.t., the reaction mixture was concentrated in vacuo. The residue was diluted
with aq.NaHCO , and extracted with CHCl . The combined organic extracts were dried over Na SO and
concentrated in vacuo. The crude product was purified by silica gel column chromatography
(hexanes/EtOAc = 1/2 to CHCl /MeOH = 97/3) to afford the amide (0.36 g, 91%). To a solution of the
C
35
H
41ClN O
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
3
3
2
4
3
amide (0.36 g, 0.45 mmol) in a 1:1 mixture of THF and MeOH (4.5 mL) was added thiourea (0.10 g, 1.34
mmol). After being stirred for 11h at 50 °C, the reaction mixture was concentrated in vacuo. The residue
was diluted with H
2
O, and extracted with CHCl
3
. The combined organic extracts were dried over Na
2
SO
4
and concentrated in vacuo. The crude product was purified by silica gel column chromatography
(CHCl
3
/MeOH = 98/2 to 97/3 to 96/4) to afford the primary alcohol (0.25 g, 76%). To a stirred solution of
i
the alcohol (0.25 g, 0.34 mmol) and AcOH (0.08 mL) in a 1:1 mixture of THF and PrOH (4.0 mL) was
added 10% Pd/C (0.20 g) under N
2
. H
2
gas was introduced and the reaction mixture was stirred under H
2
atmosphere. After being stirred for 4h at r.t., the reaction mixture was filtered through Celite and
concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl /MeOH
3
1
= 96/4 to 92/8) to afford 22 (0.17 g, 80%): H NMR (400 MHz, Chloroform-d) δ 9.45 (brs, 1H), 7.34 (d, J
=
=
8.0 Hz, 1H), 6.72 (brs, 1H), 6.17 (brs, 1H), 5.78 (d, J = 8.0 Hz, 1H), 5.60 (d, J = 1.7 Hz, 1H), 5.43 (dd, J
10.1, 3.5 Hz, 1H), 5.33 (dd, J = 3.5, 1.7 Hz, 1H), 5.20 (t, J = 10.1 Hz, 1H), 5.12 – 5.05 (m, 1H), 5.03 (dd,
J = 6.4, 1.7 Hz, 1H), 4.98 (s, 1H), 4.46 (d, J = 5.8 Hz, 1H), 4.33 (t, J = 5.1 Hz, 1H), 3.88 (dt, J = 10.1, 4.0
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Hz, 1H), 3.60 – 3.53 (m, 2H), 2.14 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 1.55 (s, 3H), 1.35 (s, 3H); C NMR
101 MHz, CDCl ) δ 170.68, 170.40, 170.28, 170.24, 163.27, 150.13, 142.81, 114.72, 102.85, 96.39, 87.34,
84.26, 80.35, 71.92, 69.35, 68.64, 66.03, 61.16, 27.25, 25.42, 20.83, 20.77, 20.74; HRMS (ESI+) m/z calcd
15 [M + H] 616.1990, found: 616.2018.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for C25
H
34
3
N O
(
2S,3S,4S)-2-((R)-2-Amino-1-((3aR,4S,6R,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-oxoethoxy)-6-(((S)-2-oxoazepan-3-yl)carbamoyl)-
3,4-dihydro-2H-pyran-3,4-diyl diacetate (23). To a stirred solution of 22 (0.17 g, 0.27 mmol) and DMSO
(
0.19 mL, 2.72 mmol) in a 5:1 mixture of CH
mmol). After being stirred for 3h at r.t., the reaction mixture was added H
a silica gel pad (CHCl
methyl-2-butene (0.5 mL) was added a solution of NaClO
g, 1.36 mmol) in H O (1.0 mL). After being stirred for 5h at r.t., the reaction was extracted with
CHCl /MeOH (9/1). The combined organic extracts were dried over Na
The crude product was purified by silica gel column chromatography (CHCl
2
Cl
2
and Et
3
N (1.4 mL) was added SO •pyridine (0.43 g, 2.72
3
2
O (0.27 mL) and passed through
t
3
/MeOH = 92/8). To a stirred solution of the crude mixture in BuOH (1.0 mL) and 2-
2
(0.12 g, 1.36 mmol) and NaH
2
PO
4
•2H O (0.21
2
2
3
2
SO
4
and concentrated in vacuo.
3
/MeOH = 9/1) to afford the
acid (0.13 g, 81%). To a stirred solution of the acid (28 mg, 0.049 mmol), 2-(S)-aminocaprolactam (24 mg,
.15 mmol), HOBt (20 mg, 0.15 mmol) and NMM (54 L, 0.49 mmol) in DMF (0.25 mL) was added EDCI
0
(47 mg, 0.25 mmol). After being stirred for 14h at r.t., the reaction was quenched with aq.NaHCO
extracted with CHCl /MeOH (9/1). The combined organic extracts were dried over Na SO
3
, and
and
3
2
4
concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl /MeOH
3
1
= 95/5) to afford 23 (30 mg, 89%): H NMR (400 MHz, Methanol-d
4
) δ 7.79 (d, J = 8.0 Hz, 1H), 5.98 (t, J
=
2.0 Hz, 1H), 5.84 (d, J = 2.8 Hz, 1H), 5.78 (d, J = 8.0 Hz, 1H), 5.62 (dd, J = 4.5, 2.6 Hz, 1H), 5.51 – 5.48
(
m, 1H), 5.45 (d, J = 3.1 Hz, 1H), 4.74 (d, J = 2.8 Hz, 1H), 4.71 (dd, J = 6.2, 2.9 Hz, 1H), 4.65 (dd, J = 6.2,
3.3 Hz, 1H), 4.59 – 4.55 (m, 2H), 3.76 – 3.67 (m, 4H), 2.09 (s, 3H), 2.04 (s, 3H), 1.86 (d, J = 13.4 Hz, 3H),
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2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
6
13
1
1
.63 – 1.52 (m, 3H), 1.51 (s, 3H), 1.45 – 1.34 (m, 3H), 1.25 (s, 3H); C NMR (101 MHz, MeOD) δ 176.35,
72.24, 171.62, 171.28, 166.24, 161.14, 151.99, 144.75, 142.76, 115.34, 106.38, 102.91, 98.75, 93.55,
87.12, 85.94, 82.02, 78.13, 67.16, 64.99, 64.46, 56.04, 53.46, 46.10, 42.52, 42.43, 32.25, 29.89, 29.08,
7.47, 25.47, 20.61, 20.52; HRMS (ESI+) m/z calcd for C29 14 [M + H] 680.2415, found: 680.2432.
2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-
dihydroxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-((S)-2-oxoazepan-3-yl)-3,4-dihydro-
H-pyran-6-carboxamide (4). A solution of 23 (30 mg, 0.044 mmol) in a 4:1 mixture of TFA and H
0.88 mL) was stirred for 4h at r.t., and concentrated in vacuo. A solution of the crude diol in a 5:1 mixture
of MeOH and Et N (0.88 mL) was stirred for 9h at r.t., filtered, and concentrated in vacuo. The crude mixture
was purified by reverse-phase HPLC [column: Luna® (C18, 10 m, 100 Å, 250 x 10 mm), solvents: 15:85
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
H
38
5
N O
(
2
2
O
(
3
MeOH:H
2
O, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 16 min] to afford DM-CAP (4, 22 mg,
) δ 7.94 (d, J = 8.1 Hz, 1H), 6.03 (d, J = 4.5 Hz, 1H), 5.85 (d, J =
1
9
0%): H NMR (400 MHz, Methanol-d
4
3.7 Hz, 1H), 5.73 (d, J = 8.2 Hz, 1H), 5.19 (d, J = 6.7 Hz, 1H), 4.68 (d, J = 1.7 Hz, 1H), 4.61 (dd, J = 11.2,
.4 Hz, 1H), 4.54 (dd, J = 5.8, 1.7 Hz, 1H), 4.36 (t, J = 4.5 Hz, 1H), 4.30 (t, J = 5.5 Hz, 1H), 4.12 (t, J = 4.4
1
Hz, 1H), 3.97 (t, J = 5.7 Hz, 1H), 3.90 (t, J = 6.5 Hz, 1H), 3.43 – 3.38 (m, 1H), 3.03 (t, J = 7.0 Hz, 1H), 2.70
– 2.64 (m, 1H), 2.07 – 1.97 (m, 2H), 1.81 (d, J = 15.2 Hz, 2H), 1.62 (q, J = 11.9, 10.7 Hz, 1H), 1.39 (s, 1H);
13C NMR (101 MHz, MeOD) δ 176.85, 174.05, 166.21, 162.02, 152.21, 144.60, 141.91, 108.72, 102.50,
1
01.60, 90.76, 84.77, 79.50, 75.68, 71.40, 63.63, 60.06, 53.44, 42.50, 31.89, 29.82, 29.14; HRMS (ESI+)
m/z calcd for C22 12 [M + H] 556.1891, found: 556.1912.
2S,3S,4S)-2-((1R)-1-((2S,5R)-4-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-
methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-((4-(4-(4-
trifluoromethoxy)phenoxy)piperidin-1-yl)benzyl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl
H
30
5
N O
(
(
diacetate (37). To a stirred solution of 32 (55 mg, 0.094 mmol), 34 (0.10 g, 0.28 mmol), HOBt (38 mg,
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0
.28 mmol) and NMM (95 L, 0.94 mmol) in DMF (0.47 mL) was added EDCI (90 mg, 0.47 mmol). After
being stirred for 7h at r.t., the reaction mixture was quenched with aq.NaHCO , and extracted with
CHCl /MeOH (9/1). The combined organic extracts were dried over Na SO and concentrated in vacuo.
/MeOH = 95/5) to afford 37
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The crude product was purified by silica gel column chromatography (CHCl
3
1
(76 mg, 87%): H NMR (400 MHz, Methanol-d
4
) δ 7.71 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.5 Hz, 2H), 7.19
(d, J = 8.8 Hz, 2H), 7.03 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.97 (t, J = 2.1 Hz, 1H), 5.91 (d, J =
8
5
.1 Hz, 1H), 5.87 (d, J = 3.8 Hz, 1H), 5.74 (dd, J = 4.5, 2.4 Hz, 1H), 5.67 (ddd, J = 4.5, 2.8, 1.7 Hz, 1H),
.43 (d, J = 2.8 Hz, 1H), 5.28 (dd, J = 5.1, 3.8 Hz, 1H), 4.70 (d, J = 1.9 Hz, 1H), 4.54 (dq, J = 7.5, 3.8 Hz,
1H), 4.42 (d, J = 14.5 Hz, 1H), 4.38 (dd, J = 6.2, 1.9 Hz, 1H), 4.33 (d, J = 14.4 Hz, 1H), 3.82 – 3.79 (m,
1
3
1
H), 3.48 (td, J = 9.0, 6.8, 3.5 Hz, 3H), 3.13 – 3.06 (m, 2H), 3.05 (s, 3H), 2.09 (s, 3H), 2.09 (s, 3H), 2.06 (s,
13
H), 1.87 (dtd, J = 12.3, 8.2, 3.6 Hz, 3H); C NMR (101 MHz, MeOD) δ 172.85, 171.84, 171.43, 171.17,
66.07, 162.39, 157.61, 152.11, 151.92, 144.94, 143.92, 141.57, 130.89, 130.02 (2C), 123.59 (2C), 118.04
(2C), 117.98 (2C), 106.19, 103.75, 98.26, 89.36, 83.17, 78.98, 76.60, 74.71, 73.98, 64.75, 59.35, 48.18,
8.16, 31.43 (2C), 20.70, 20.52, 20.49; HRMS (ESI+) m/z calcd for C42 16 [M + H] 934.2970,
found 934.2998.
4
H
47
F
3
5
N O
(2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-3-
methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-(4-(4-(4-
(
trifluoromethoxy)phenoxy)piperidin-1-yl)benzyl)-3,4-dihydro-2H-pyran-6-carboxamide (5).
solution of 37 (76 mg, 0.082 mmol) in a 5:1 mixture of MeOH and Et N (1.6 mL) was stirred for 6h at r.t.,
and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18,
A
3
1
0 m, 100 Å, 250 x 10 mm), solvents: 65:35 MeOH:H
2
O, flow rate: 3.0 mL/min, UV: 254 nm, retention
) δ 7.86 (d, J = 8.1 Hz,
1
time: 18 min] to afford CPPB (5, 63 mg, 95%): H NMR (400 MHz, Methanol-d
4
1
H), 7.20 (dd, J = 10.9, 8.6 Hz, 4H), 7.02 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.98 (dd, J = 3.3,
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6
1
1
.0 Hz, 1H), 5.80 (d, J = 4.4 Hz, 1H), 5.76 (d, J = 8.2 Hz, 1H), 5.21 (d, J = 4.6 Hz, 1H), 4.68 (d, J = 2.0 Hz,
H), 4.54 (dp, J = 7.3, 3.6 Hz, 1H), 4.48 (dd, J = 5.2, 2.0 Hz, 1H), 4.44 (d, J = 14.5 Hz, 1H), 4.39 (t, J = 3.9
Hz, 1H), 4.33 (d, J = 14.6 Hz, 1H), 4.20 (t, J = 4.7 Hz, 1H), 4.06 – 4.02 (m, 1H), 3.66 (t, J = 5.1 Hz, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
1
7
.52 – 3.45 (m, 3H), 3.19 (s, 3H), 3.08 (ddd, J = 12.3, 8.6, 3.4 Hz, 3H), 2.15 – 2.07 (m, 2H), 1.86 (dtd, J =
13
2.2, 8.3, 3.5 Hz, 3H); C NMR (101 MHz, MeOD) δ 173.68, 166.16, 163.31, 157.62, 152.18, 152.08,
44.15, 141.83, 130.91, 129.76 (2C), 123.58 (2C), 118.03 (4C), 109.58, 102.74, 100.64, 90.83, 83.32, 80.35,
7.51, 74.27, 74.01, 67.61, 63.48, 58.61, 48.20 (2C), 43.51, 31.47 (2C); HRMS (ESI+) m/z calcd for
C
36
H
41
F
3
N
5
O
13 [M + H] 808.2653, found 808.2675.
2S,3S,4S)-2-((1R)-1-((2S,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-((4-(4-(4-
trifluoromethoxy)phenoxy)piperidin-1-yl)benzyl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl
(
(
diacetate (35). To a stirred solution of 18 (33 mg, 0.056 mmol), 34 (62 mg, 0.17 mmol), HOBt (23 mg,
0.17 mmol) and NMM (62 L, 0.56 mmol) in DMF (0.28 mL) was added EDCI (54 mg, 0.28 mmol). After
being stirred for 14h at r.t., the reaction was quenched with aq.NaHCO
3
, and extracted with CHCl /MeOH
3
(
9/1). The combined organic extracts were dried over Na
was purified by silica gel column chromatography (CHCl
NMR (400 MHz, Methanol-d
2
SO
4
and concentrated in vacuo. The crude product
1
3
/MeOH = 95/5) to afford 35 (43 mg, 82%): H
4
) δ 7.69 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H),
7
.02 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 5.99 – 5.97 (m, 1H), 5.95 (d, J = 5.8 Hz, 1H), 5.91 (d, J
=
8.1 Hz, 1H), 5.74 – 5.71 (m, 1H), 5.66 – 5.63 (m, 1H), 5.46 (d, J = 3.2 Hz, 1H), 5.05 (t, J = 4.7 Hz, 1H),
4
1
.78 (d, J = 2.0 Hz, 1H), 4.57 – 4.52 (m, 1H), 4.49 (d, J = 14.5 Hz, 1H), 4.39 – 4.34 (m, 1H), 4.30 (d, J =
4.7 Hz, 1H), 3.84 – 3.80 (m, 1H), 3.64 (s, 3H), 3.53 – 3.45 (m, 2H), 3.13 – 3.06 (m, 2H), 2.83 (t, J = 6.5
1
3
Hz, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 1.87 (dtd, J = 12.7, 8.5, 3.5 Hz, 3H); C NMR (101 MHz,
MeOD) δ 172.92, 171.74, 171.36, 170.39, 166.18, 162.23, 157.56, 152.06, 151.81, 145.05, 140.90, 130.78,
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Page 29 of 74
Journal of Medicinal Chemistry
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29.96 (2C), 123.52 (2C), 117.96 (2C), 117.92 (2C), 105.75, 103.47, 98.22, 88.92, 82.72, 78.21, 76.44,
3.95, 67.92, 64.42, 61.21, 58.65, 58.20, 48.10 (2C), 43.57, 31.38 (2C), 20.60, 20.44, 20.39; HRMS (ESI+)
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m/z calcd for C42
2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-
methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-(4-(4-(4-
trifluoromethoxy)phenoxy)piperidin-1-yl)benzyl)-3,4-dihydro-2H-pyran-6-carboxamide (6).
solution of 35 (43 mg, 0.046 mmol) in a 5:1 mixture of MeOH and Et N (0.92 mL) was stirred for 6h at r.t.,
and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18,
H
47
F
3
N
5
O16 [M + H] 934.2970, found 934.2991.
0
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7
8
9
0
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0
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0
(
(
A
3
1
0 m, 100 Å, 250 x 10 mm), solvents: 65:35 MeOH:H
2
O, flow rate: 3.0 mL/min, UV: 254 nm, retention
) δ 7.79 (d, J = 8.2 Hz,
1
time: 19 min] to afford I-CPPB (6, 35 mg, 93%): H NMR (400 MHz, Methanol-d
4
1
5
H), 7.19 (d, J = 8.6 Hz, 4H), 7.02 (d, J = 9.1 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 5.98 (d, J = 3.6 Hz, 1H),
.94 (d, J = 5.1 Hz, 1H), 5.77 (d, J = 8.0 Hz, 1H), 5.36 (d, J = 5.8 Hz, 1H), 4.67 – 4.65 (m, 1H), 4.61 (d, J
= 2.8 Hz, 1H), 4.48 (dd, J = 8.6, 4.6 Hz, 1H), 4.36 – 4.33 (m, 1H), 4.27 (dd, J = 5.4, 2.7 Hz, 1H), 3.71 –
.65 (m, 2H), 3.51 (d, J = 4.4 Hz, 2H), 3.48 (s, 3H), 3.25 – 3.18 (m, 2H), 2.13 – 2.00 (m, 3H), 1.92 – 1.76
3
1
3
(
m, 3H); C NMR (101 MHz, MeOD) δ 173.68, 166.15, 163.30, 157.62, 152.18, 152.08, 144.15, 141.83,
130.90, 129.76 (2C), 123.58 (2C), 118.10 (2C), 118.02 (2C), 109.57, 102.73, 100.63, 90.83, 83.32, 80.35,
7.51, 74.27, 74.01, 67.61, 63.48, 58.61, 48.20 (2C), 43.51, 31.47 (2C); HRMS (ESI+) m/z calcd for
13 [M + H] 808.2653, found 808.2668.
(2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-
7
C
36
H
41
F
3
5
N O
dimethoxytetrahydrofuran-2-yl)-2-oxoethoxy)-6-((4-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1-
yl)benzyl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (38). To a stirred solution of 33 (38 mg,
0
.068 mmol), 34 (75 mg, 0.20 mmol), HOBt (28 mg, 0.20 mmol) and NMM (75 L, 0.68 mmol) in DMF
(0.34 mL) was added EDCI (65 mg, 0.34 mmol). After being stirred for 7h at r.t., the reaction was quenched
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