Potent and Specific Inhibition of NTCP-Mediated HBV/HDV Infection and Substrate Transporting by a Novel, Oral-Available Cyclosporine A Analogue

Article abstract of DOI:10.1021/acs.jmedchem.0c01484

Analogues of the natural product cyclosporine A (CsA) were developed and assessed as antivirals against infection of hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV). An analogue termed 27A exhibits potent inhibition of HBV/HDV infection by specifically blocking viral engagement to its cellular receptor NTCP, while it lacks immunosuppressive activity found in natural CsA. Intraperitoneal injection or oral intake of 27A protects HDV-susceptible mouse model from HDV infection. 27A serves as a promising lead for the development of novel anti-HDV/HBV agents.

Full text of DOI:10.1021/acs.jmedchem.0c01484

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Article
Potent and Specific Inhibition of NTCP-Mediated HBV/HDV Infection
and Substrate Transporting by a Novel, Oral-Available Cyclosporine
A Analogue
Yang Liu, Hanying Ruan, Ying Li, Guoliang Sun, Xiao Liu, Wenhui He, Fengfeng Mao, Miaomiao He,
Liwei Yan, Guocai Zhong, Huan Yan, Wenhui Li,* and Zhiyuan Zhang*
Cite This: J. Med. Chem. 2021, 64, 543−565
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ABSTRACT: Analogues of the natural product cyclosporine A (CsA) were developed and assessed as antivirals against infection of
hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV). An analogue termed 27A exhibits potent inhibition of HBV/HDV
infection by specifically blocking viral engagement to its cellular receptor NTCP, while it lacks immunosuppressive activity found in
natural CsA. Intraperitoneal injection or oral intake of 27A protects HDV-susceptible mouse model from HDV infection. 27A serves
as a promising lead for the development of novel anti-HDV/HBV agents.
NTCP, and their derivatives were demonstrated to reduce Pre-
S1-NTCP binding and viral infection.10,12,13 Furthermore,
NTCP variant rs2296651 (Ser267Phe), which loses taurocho-
late transport activity and the ability to support HBV infection
in vitro,12 was reported to be inversely associated with chronic
hepatitis B and progression to cirrhosis and hepatocellular
carcinoma.14 Collectively, these studies indicate that NTCP is
a promising therapeutic target for antivirals against the viral
infection.
To date, several classes of small-molecular inhibitors
targeting NTCP have been identified. A structure−activity
relationship study using computational analysis and pharma-
cophore characteristics of NTCP substrates estimated 31 FDA-
approved drugs as potential NTCP inhibitors.15 Some of those
drugs, including cyclosporine A,16,17 irbesartan, ritonavir,18 and
INTRODUCTION
■
Hepatitis B virus (HBV) infection remains a major global
public health problem; chronic HBV infection and related
diseases cause around 1 million deaths annually worldwide.1
HDV is a satellite virus of HBV and replicates only in the
presence of envelope proteins of HBV.2,3 Co-infection of HBV
with HDV represents the most severe viral hepatitis.4
Identification of sodium taurocholate co-transporting peptide
(NTCP, encoded by the SLC10A1 gene) as a cellular receptor
for both HBV and HDV significantly advanced our under-
standing of the viral infections.5 NTCP is a bile acid
transporter that predominantly expressed on the sinusoidal
membrane of hepatocytes, and its expression is subjected to
complicated regulation of liver-specific transcription factors
and microenvironment.6,7 NTCP-mediated HBV infection
initiates from the specific interaction between the HBV Pre-
S1 domain of the large envelope protein (L) and NTCP.5,8,9 A
small motif of NTCP-containing residues 157−165 is essential
for HBV Pre-S1 binding, and it is responsible for the species
specificity of HBV infection between human and nonhuman
primates.5,10,11 Moreover, residues required for NTCP uptake
of its substrates are also employed by HBV and HDV entry.12
Consistent with this, bile acids, the natural substrates of
Received: August 26, 2020
Published: December 28, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
© 2020 American Chemical Society
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a
Scheme 1. Synthesis of Compounds 3, 4, 7, and 8
a
Reagents and conditions: (a) O3, Me2S, dichloromethane (DCM), −78 °C, 30 min; (b) MeOH, p-TsOH, room temperature (RT), 16 h; (c)
NaBH4, MeOH, RT, 16 h; (d) MsCl, triethylamine (TEA), DCM, 0 °C then RT, overnight; (e) Jones reagent, acetone, 0 °C then RT, overnight;
(f) t-BuOK, methyltriphenylphosphonium bromide, tetrahydrofuran (THF), −78 °C then 0 °C, 3 h; (g) 10% Pd/C, MeOH, H2, 1 atm, RT, 16 h.
a
Scheme 2. Synthesis of Compounds 10−52
a
Reagents and conditions: (a) acetic anhydride (Ac2O), pyridine (Py), RT, overnight; (b) appropriate mercapto heterocyclic and aromatic,
trimethylsilyl trifluoromethanesulfonate (TMSOTf), N,O-bis(trimethylsilyl)acetamide (BSA), acetonitrile (ACN), 50 °C, 90 min; (c) methyl 2-
hydroxyacetate or methyl 3-hydroxypropanoate, silver trifluoromethanesulfonate, activated 3 Å molecular sieves, DCM, RT, 2 h; NIS, activated 3 Å
molecular sieves, ACN, RT, 2 h; (d) 1 N NaOH, MeOH, RT, 4.5 h; (e) 3-(benzyloxy)phenol or 3,5-dimethoxyphenol, FeCl3, 3 Å molecular sieves,
DCM, RT, 16 h.
ezetimibe,18,19 have been tested for their activity against HBV/
HDV infection on cell cultures. Bile acid derivatives and
cholesterol derivatives10,12,13,20 that have similar structures to
substrates of NTCP were also reported as HBV entry
inhibitors. However, none of these compounds were tested
in vivo against HBV.21 Myrcludex B, the first-in-class and only
NTCP inhibitor under clinical development, is a synthetic
lipopeptide derived from the Pre-S1 domain of the HBV
envelope protein.22,23 Its safety and effectiveness in patients co-
infected with HBV and HDV have been confirmed in Phase II
clinical trial.24 However, Myrcludex B needs to be adminis-
trated via subcutaneous injection, developing novel and oral-
available NTCP inhibitor is desirable.
as an immunosuppressive drug for organ transplantation
patients. Previous work has shown that CsA inhibits bile
acids uptake by NTCP26,27 and NTCP-mediated HBV and
HDV entry.16,17 In this study, we report the synthesis of a large
series of CsA analogues. We also used NTCP-complemented
HepG2 cells, which are susceptible to HBV and HDV
infection, to assess the antiviral activities of these analogues
using in vitro HBV/HDV infection assays. We ultimately
obtained a highly potent and very specific compound that
targets NTCP, and we used multiple approaches to assess its
antiviral mechanism and to verify its activity in vivo.
RESULTS AND DISCUSSION
■
Cyclosporine A (CsA) is a cyclic peptide comprising 11
amino acids that was originally isolated from the ascomycete
fungus Tolypocladium inflatum.25 It is widely used in the clinic
Chemistry. The synthesis route for the five-membered ring
series of CsA analogues is depicted in Schemes 1,2 and 2. The
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Article
Figure 1. Strategy for developing NTCP inhibitor from CsA.
Table 1. SAR of Alkyl Substitutions at the C-5 Position of the Five-Membered Ring of CsA Derivative
a
a
compd
A
R
IC50 (μM)
compd
A
R
IC50 (μM)
>10
>10
>10
>10
CsA
2
3
11
12
13
5.784 1.122
11.913 1.914
13.504 8.126
>10
9.260 1.952
>10
4
6
7
8
C
C
C
C
H
O
O
O
O
O
H
Me
ethan-1-one
prop-1-en-2-yl
i-Pr
methyl acetate
methyl propionate
propionic acid
10A
0.281 0.149
0.523 0.260
b
10m (1:3.3)
a
b
HBV infection of HepG2-NTCP cells assay. Mean values from a minimum of three experiments. A mixture of two isomers. Ratio of main
product to minor product (A/B).
olefinic bond of CsA 1 was broken down to form an aldehyde
by ozone, which then self-cyclized to give compound 2. A
methylated product compound 3 was synthesized from
compound 2 by carrying out a reaction in methanol in the
presence of p-toluenesulfonic acid. Another analogue com-
pound 4 was prepared through three steps: reduction of
hemiacetal 2 in methanol in the presence of reducing reagent
NaBH4 gave the ring-opening product compound 5, which was
then reacted with MsCl and triethylamine (TEA) and
instinctively self-cyclized to give target compound 4. The
ketone analogue 6 was obtained by treating compound 1 with
Jones reagent. Compound 6 was converted to compound 7 via
a Witting reaction. Hydrogenation of compound 7 using 10%
Pd/C in MeOH gave compound 8.
methyl 2-hydroxyacetate or methyl 3-hydroxypropanoate,
respectively. Hydrolysis of 12 gave the acid derivative 13.
Other O-linked CsA analogue compounds 43 and 44 were
prepared via treating compound 2 with 3,5-dimethoxyphenol
or 3-(benzyloxy) phenol catalyzed by Lewis acid FeCl3 in the
presence of dry 3 Å molecular sieves. Both of isomers 43A and
43B were separated through diligent chromatography.
Unfortunately, we could not distinguish the α or β isomers
from the NMR-1H spectrum. Attempts of characterization by
X-ray crystal structure to find the right configuration failed, as
we could not obtain either crystal form of two isomers.
Rational Design of an NTCP Inhibitor. Since NTCP was
discovered as the key receptor for HBV and HDV infection, a
few research groups have reported a number of small-molecule
NTCP inhibitors, such as bile acid analogues and CsA
derivatives,16,17 that are able to block virus infection. In our
hands, the natural product CsA showed inhibition at IC50 of
5.8 and 1 μM against HBV and HDV infection in cell assays,
respectively, indicating a good starting hit to continue
medicinal chemist effort to improve its potency. We noted
that previously reported CsA-based structures, which were
generated by replacing or modifying some residues on the
cyclic peptide macrocycle, did not have much potency
improvement, even though intensive effort had been made
toward compound synthesis. Our attention then focused on
the unusual butenyl-methyl-threonine (BMT) amino acid as it
has two workable (olefin and hydroxyl) functional groups for
further derivatizing.
The synthetic routes for compounds 10−52 are illustrated in
Scheme 2. We started from preparing a common intermediate
compound 9 by acetylation of compound 2 with acetic
anhydride in pyridine. The designed five-membered ring
analogues of CsA, compound 10, and compounds 14−42
and 45−52 were synthesized by treating the intermediate 9
with various mercapto heterocyclics and mercapto aromatics
together with trimethylsilyl trifluoromethanesulfonate
(TMSOTf) and N,O-bis(trimethylsilyl)acetamide (BSA) in
acetonitrile (CH3CN), as a mixture of α,β racemic. Normally,
the major product (isoform A) could be separated and defined
by a typical double-doublets peak with J-coupling constants of
1
6.4 and 8.8 Hz around δ 4.2−4.4 ppm in the H NMR
spectrum as a character signal. The isoform B could be isolated
as a minor product (dd, J = 6.0, 8.0 Hz, δ around 4.0−4.15 Hz)
occasionally.
As we have little structure−activity relationship (SAR)
information about CsA against HBV and HDV interaction with
NTCP, we started with design and synthesis of a few long-
chain analogues by manipulating the olefin functional group.
Compound 10 was also used as a starting material for the
synthesis of the O-linked compounds 11−12 by reacting with
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Table 2. Optimizing the Monosubstituted Benzene Ring in the Structure of Compound 10
a
a
a
compd
IC50 (μM)
compd
23A
IC50 (μM)
compd
IC50 (μM)
14B
15A
16A
16B
17A
18A
19A
20A
21A
22A
5.329 1.053
4.955 0.952
0.990 0.331
1.700 0.960
0.262 0.148
1.541 0.316
1.266 0.355
0.256 0.026
1.650 1.320
0.530 0.152
0.181 0.045
0.223 0.038
0.365 0.066
0.249 0.082
0.110 0.015
0.013 0.002
0.172 0.039
0.354 0.038
0.317 0.075
0.261 0.077
31A
31m (1:1.5)
0.269 0.073
0.253 0.096
0.382 0.079
0.480 0.004
1.199 0.554
0.206 0.061
0.171 0.152
0.344 0.058
0.681 0.097
b
24A
25A
26A
27A
27B
28A
29A
30A
32A
33A
34A
35A
36A
43A
43B
b
30m (1:1)
a
b
HBV infection of HepG2-NTCP cells assay. Mean values from a minimum of three experiments. A mixture of two isomers. Ratio of main
product to minor product (A/B, A is the α-isoform, and B is the β-isoform).
However, these analogues did not show any potency
improvement at all (data not shown). Having been inspired
by sugar structures and chemistry, we came up with an idea of
five-membered ring structure that would give us a larger
working space for expending the medicinal chemistry effort.
Following the design strategy outlined in Figure 1, we
initialized our synthetic efforts based on a scaffold-hopping
approach by breaking the carbon−carbon double bond to form
a hemiacetal ring system and generate the bicyclic skeleton. A
first set of testing compounds, compounds 2−13, were
synthesized (Table 1), while hemiacetal 2 and thioglycoside
10 were common intermediates used for the synthesis of S-
glycoside and O-glycoside analogues, while C-glycoside
analogues were synthesized starting from original CsA. All
newly synthesized compounds were tested in HBV infection
assays with the HepG2-NTCP cells to determine the IC50 of
their antiviral activity. Surprisingly, all compounds of the first
set of cyclic CsA analogues did not show good inhibition even
at a 10 μM compound concentration with an exception that p-
thiocreasol 10A showed IC50 at 281 nM, which is a 20-fold
improvement compared to the initial hit CsA. According to the
encouraging result, compound 10 seems to be a decent hit that
sets up a new direction for further SAR development.
removing the methyl group on phenyl ring from compound 10,
had sharply decreased inhibition activity (IC50 = 5.3 μM).
Similar activity was observed on the pyrimidyl compound 15A
(IC50 = 5.0 μM). However, a pair of separated racemic 4-
methoxyl compounds 16A and 16B also lost 3.5-fold and 6-
fold activity, respectively, indicating that α-isomer and β-
isomer might follow a different SAR. 4-Butyl compound 17A,
extending the carbon chain at 4-position, maintained its
inhibitory activity. Substitution with polar functional groups
like methylpiperazine (compound 18A) and morpholine
(compound 19A) also caused 5.5-fold and 4.5-fold activity
loss. Interestingly, the 4-O-benzyl compound 20A maintained
the inhibition activity at the same level as compound 10A,
which indicates that the extended phenyl group might form a
new interaction with the binding protein.
To investigate whether the substituent at the C-3 position
affects anti-HBV activity, we synthesized compounds 21A−
26A, 27A, 27B, 28A, and 29A (Table 2). Compound 21A,
with a hydroxyl group introduced at the C-3 position, was 3.2-
fold more potent than the original phenyl compound 14B. The
extension of the side chain from the 4-oxygen atom resulted in
a notable increase in potency based on inhibition testing
results, such as compound 22A (IC50 = 0.530 μM, 3-fold vs
21A), compound 23A (IC50 = 0.181 μM, 9-fold vs 21A) and
compound 24A (IC50 = 0.223 μM, 7.4-fold vs 21A). Further
extending the carbon chain or adding a larger alkyl group did
not further improve the anti-HBV infection activity (25A−
26A, 28A−29A). One pair of 4-O-benzyl compound 27A and
27B gave the best inhibition activities. The isomeric activity of
4-O-benzyl compounds 27A gave anti-HBV infection power at
an IC50 of 0.110 μM. Surprisingly, another isolated isomer 27B
Lead Optimization. On the basis of the above findings,
considering synthesis accessibility, we deployed a design
strategy focusing on further modifications on the mercapto
aromatic ring of compound 10. Therefore, compound 10A
(IC50 = 0.281 μM) was used as a starting point for extending
the structure−activity relationship (SAR) studies. We first
explored a set of compounds to obtain an SAR on the 4-
position of the phenyl ring (Table 2). Compound 14B,
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showed excellent inhibition potency with IC50 = 0.013 μM, 9-
fold more potent than compound 27A. The pyridinyl
analogues 30A, 31A, 32A, and 3-N-methylpyridone 33A
were slightly less active than 27A; however, those B isoforms
seemed having similar potency to their A isoforms based on
the inhibition results of the mixtures of 30m and 31m. In
addition, we found that 3-N-substituted compounds (34A−
36A) had slightly reduced inhibition activity compared to the
3-O-substituted compounds (22A-24A). Even though com-
pound 27B showed the profound inhibition potency, many
reaction methods had been tried to reverse the α,β selectivity
during the synthesis of 27B isomer. Unfortunately, the B
isoform was always 10 times less than the A isoform in the
mixture and difficult to be separated. We have devoted much
effort toward crystallization of these isomers to identify their
conformation, but we did not obtain any crystal. We then
conducted a two-dimensional (2D) NMR study on 27A and
27B. The data from a series of 2D NMR experiments seem to
support β-configuration for compound 27B as we see long-
distance coupling between the Me group on the five-
membered ring with the anomeric hydrogen Ha, also coupling
between the anomeric hydrogen Ha and the hydrogen Hb on
the five-membered ring. Instead, from the NOE data of
compound 27A, we did not see the long-distance coupling
between the Me group and Ha, and the coupling between Ha
and Hb (see Supplemental Figure 1). These data may help
denote 27A as the α-isoform and 27B as the β-isoform. This
general pattern should apply to other compounds, A isoform
should be the α-isoform and B isoform should be the β-
isoform.
angle change along the C−S−Ar bonds. We next synthesized a
few O-glycoside compounds (43A, 43B, and 44A) to compare
with their S-glycoside analogues (27A, 27B, and 38A). The O-
glycoside compounds had about 3- to 5-fold activity loss
compared with their S-glycoside analogues, indicating that the
S-glycoside analogues may be better inhibitors for anti-HBV
infection.
Building from these SAR findings, we further focused on
halogen modification on the second benzyl ring of 27A (45A−
50A), hoping to get structures with improved potency or
metabolic stability (Table 4). Unfortunately, no significant
Table 4. Further Optimization of Compound 27A
a
compound
45A
R1
R
IC50 (μM)
H
H
H
H
H
H
Cl
MeO
MeO
2-F
3-F
4-F
0.318 0.104
0.197 0.029
0.206 0.050
0.403 0.195
0.335 0.111
0.122 0.030
0.417 0.112
0.216 0.082
0.132 0.004
46A
47A
48A
49A
50A
51A
52A
2-Cl, 4-F
3-Cl, 4-F
2-Me, 4-F
4-F
4-F
4-F
b
52m (1:1.4)
a
To further investigate whether the double-substituent
benzene ring would affect the anti-HBV infection activity, a
set of disubstituted compounds were synthesized and tested via
anti-HBV infection assay (Table 3). Compared with the 3-
HBV infection of HepG2-NTCP cells assay. Mean values from a
b
minimum of three experiments. A mixture of two isomers. Ratio of
main product to minor product (A/B).
activity improvement was achieved. These compounds
retained similar or slightly decreased activity compared to
compound 27A (Table 4). A slight modification on both
phenyl rings (compounds 51A and 52A) did not improve the
potency either, indicating little opportunity to achieve further
enhancement on the potency. Having successfully improved
the potency from the low micromolar initial hit CsA to a low
nanomolar compound based on HBV infection assays with
HepG2-NTCP cells, we next assessed metabolic stability of
selected compounds (Table 5). According to their liver
microsomal stability, compounds 27A, 27B, 32A, and 47A
Table 3. Optimizing the Disubstituted Benzene Ring in the
Structure of Compound 10
a
compd
R
IC50 (μM)
37A
38A
39A
40A
41A
42A
44A
3-OMe, 4-OMe
3-OMe, 5-OMe
3-Cl, 5-OMe
3-OMe, 4-Cl
3-O-Bn, 4-Cl
2-OMe, 6-OMe
0.346 0.069
0.117 0.042
0.116 0.059
0.101 0.057
0.215 0.006
2.169 0.832
0.689 0.340
Table 5. Human/Rat/Mouse Liver Microsome Stability
Detection
a
a
a
compound
HLM CLint
RLM CLint
MLM CLint
17A
23A
27A
27B
36A
38A
39A
40A
47A
50A
6.1
18.5
8.0
7.7
6.1
10.2
4.2
14.8
7.7
4.0
2.3
2.2
3.0
1.4
0.7
4.9
2.2
2.0
0.8
2.5
4.0
4.1
2.1
2.1
5.5
2.2
3.3
0.3
7.1
a
HBV infection of HepG2-NTCP cells assay. Mean values from a
minimum of three experiments.
methoxyl parent compound 22A (IC50 = 0.530 μM), the 3,4-
di-OMe compound 37A (IC50 = 0.346 μM) had a slight
potency increase; however, the 3,5-di-OMe compound 38A
(IC50 = 0.117 μM), 3-Cl-5-OMe compound 39A (IC50 = 0.116
μM), and 3-OMe-4-Cl compound 40A (IC50 = 0.101 μM)
showed 5-fold activity enhancement. Compound 41A, the 3-
OBn-4-Cl analogue, had about 2-fold activity increase.
Surprisingly, the 2,6-di-OMe compound 42A dramatically
lost the inhibition potency, presumably owing to a torsional
13.0
a
CLint: μL/(min mg protein), determined in human/rat/mouse
microsomes.
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were selected for basic pharmacokinetics (PK) analysis in a
preliminary oral mouse PK study, while plasma samples were
collected and tested at 8 and 24 h time points to monitor the
drug concentration (Table S1; Supporting Information). The
plasma protein binding (PPB) of compound 27A in mouse and
human was detected (Table S2; Supporting Information).
Overall, compound 27A exhibited the most attractive
combination of better potency and PK profile; therefore, it
was selected for full PK profiling study, which gave a very long
half-life in mouse plasma and good bioavailability in both oral
administration or IP dosing (Table 6); thereafter, it was
selected as a potential candidate for further evaluation.
injection: the mice in group A were injected 27A at dose of 40
mg/kg before HDV challenge (n = 4); the mice in group B
were dosed 27A at 40 mg/kg for two consecutive days (n = 4);
and the mice in group C were dosed with vehicle (n = 5). All
of the mice in each group were challenged with HDV virus at a
dose of 5 × 109 genome equivalents and then sacrificed on the
7th day to determine the virus infection efficiency. Group A
with a single dose of 27A given before HDV infection
significantly decreased the HDV RNA levels in the liver
compared to the vehicle control (n = 4). Group B with an
additional dose of 27A further enhanced its antiviral efficacy (n
= 4) (Figure 3B). As compound 27A was designed as an oral
drug, we wanted to know if oral administration would also
work well. Given that adult hNTCP-tg mice are not susceptible
to HDV infection, while oral administration to neonatal mice is
not easy to conduct, we decided to orally dose compound 27A
to adult HDV-susceptible mice model: the NTCP-edited FVB
mice that was previously reported.30 The results, shown in
Figure 3C, indicated that an oral gavage dose of 30 mg/kg 27A
prior to HDV infection efficiently decreased the HDV RNA
levels in the liver (n = 4) compared to the vehicle control
group (n = 4) (Figure 3C). Collectively, these in vivo study
results revealed that both intraperitoneal injection and oral
administration of compound 27A can achieve promising
antiviral efficacy.
Table 6. Pharmacokinetic Parameters for Intravenous (IV),
Intraperitoneal (IP), and Oral (PO) Administration of 27A
in C57BL/6 Mice
AUCInf
(h*ng/mL)
tmax
Cmax
CL
F
route
t1/2 (h) (h) (ng/mL) (mL/min/kg) (%)
a
IV
IP
39 673
87 351
14 518
6.027
5.683
5.651
2.1
b
2
1
12399
2287
110
18
c
PO
a
b
c
IV dose: 5 mg/kg. IP dose: 10 mg/kg. PO dose: 10 mg/kg. All
administrated with dimethyl sulfoxide (DMSO):cremophor:0.9%
NaCl = 5:5:90. Values were the mean values of male for mouse (n
= 3).
Mode-of-Action Study. Encouraged by the good results of
in vivo efficacy studies, we then moved to investigate the mode
of action of compound 27A to understand how it interrupts
the HBV infection process. Considering NTCP on the
hepatocyte cell surface is the key receptor protein utilized by
the virus for entry into cell causing infection, whereas CsA is
known to inhibit NTCP function, we assessed that 27A may
act as a blocker to prevent the initial interaction between virus
and NTCP by sitting on a certain site or act as a modulator to
interfere with NTCP surface localization. During our in vivo
animal efficacy study, we have observed that the taurocholic
acid level in the mice was increased (Supplement Figure 4).
Given that NTCP is a cholic acid transporter, over all of these
observations, it is implied that NTCP seems to be a
Antiviral Activity. After 27A being selected as an advanced
compound, we then carried out more detailed antiviral activity
tests around compound 27A. We tested 27A on primary
human hepatocytes (PHH) and found that 27A could inhibit
HBV infection on PHH cells (estimated IC50 = 59 nM)
(Supplement Figure 2). We then found that 27A could reduce
the infection of HBV genotypes B and C (Figure 2A), which
differ in sequence from the genotype D virus strain used in the
IC50 determination. These results indicate that 27A exerts pan-
genotypic anti-HBV effects. We next investigated the anti-HBV
mechanism of 27A. Southern blotting of HBV DNA, which
was extracted from infected HepG2-NTCP cells, indicated that
27A could significantly decrease the HBV cccDNA level
compared to the DMSO control upon addition of the HBV
inoculum, in a dose-dependent manner (Figure 2B).
Consistent with this, viral transcription was also dramatically
decreased by 27A, as revealed in a Northern blot assay
targeting HBV RNAs (Figure 2C, left and middle panels). In
contrast, when 27A was added on day 3 post infection, the
transcription level of HBV virus was not affected (Figure 2C,
right panel), implying that 27A inhibits HBV infection at an
early stage of HBV life cycle.
In addition, 27A inhibits the infection of Hepatitis D virus,
which is an accepted surrogate for HBV entry into HepG2-
NTCP cells.3,4,28 Specifically, treatment with 200 nM 27A
significantly decreased the infection ratio of HDV to 50% of
the DMSO control, with the decrease rising to over 95% at a
concentration of 5 μM, in a dose-dependent manner (Figure
2D). Collectively, these results indicate that 27A blocks HBV
and HDV entry into hepatocytes, demonstrating the strong
potential of 27A as an anti-HBV and HDV agent with
apparently attractive potency.
3
suspectable target of 27A. Therefore, we tested 27A in H-
taurocholic acid (3H-TCA) uptake assays using HepG2-NTCP
cells. 27A can inhibit NTCP-mediated uptake of its substrate
3H-TCA with an IC50 value of 0.066 0.031 μM (Figure 4A),
similar to the IC50 value of 27A in the inhibition of HBV
infection assay, but showed no cytotoxicity at 100 μM. This
result supported that the antiviral activity of 27A may associate
with the inhibition of NTCP function.
It is known that HepG2-NTCP cells under treatment with
taurolithocholic acid (TLCA) can induce NTCP endocyto-
sis.12 We want to know if 27A plays the same action or through
a different mechanism. We then measured NTCP distribution
by performing immunostaining of NTCP after treating the
HepG2-NTCP cells with 27A or without. The cells treated
with 27A showed a similar surface expression level of NTCP to
cells treated with DMSO, whereas the cells treated with
taurolithocholic acid (TLCA) showed decreased NTCP
staining intensity at the cell surface (Figure 4B). These results
clearly indicated that the mode of action of 27A is through
blocking taurocholic acid transporting function of NTCP
function rather than through inducing NTCP endocytosis.
We next used Myr59F, an FITC-labeled polypeptide derived
from HBV Pre-S1 polypeptide used as a surrogative indicator,
to visualize the binding between HBV and NTCP of HepG2-
Having investigated the in vitro efficacy of 27A, we then
focused on verifying its antiviral efficiency in vivo. hNTCP-tg
mice, which are susceptible to HDV infection,29 were divided
into three groups and administrated through intraperitoneal
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Figure 2. Antiviral activity of 27A. (A) 27A inhibits genotype B and genotype C HBV virus infection of HepG2-NTCP cells. HBeAg and HBsAg
titers that secreted in the medium were determined at dpi 5 by commercial ELISA kits. The concentration of the compounds used in the assay was
2 μM for 27A and 200 nM for Myr47. (B) Southern blot assay on HBV DNA extracted from HBV-infected HepG2-NTCP cells. HBV DNA was
extracted on dpi 5 by Hirt methods. Southern blotting was conducted with DIG-labeled probe that targeting full-length HBV genome (DIG-high
prime DNA-labeling and detection starter kit II, Roche, cat. No. 11585614910). (C) Northern blot assay on RNAs extracted from HBV-infected
HepG2-NTCP cells. 27A was added to the cells with the inoculum of the virus (lanes 3 and 4) or at dpi 3−5 (lanes 5 and 6). The blotting was
procedure was similar to that of Southern blot except for the additional DIG-labeled glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe.
(D) 27A inhibits HDV infection of HepG2-NTCP cells. HDV delta antigen was stained by a homemade FITC-labeled antidelta antigen antibody
at dpi 5. The pictures were captured by an Opera High Content Screening System (PerkinElmer). Blue: 4′,6-diamidino-2-phenylindole (DAPI)
stain, green: HDV delta antigen. (E) HDV infection ratio calculation of (D) was conducted by Columbus Image Analysis System (PerkinElmer).
NTCP cells. Myr47, a nonlabeled Pre-S1-derived peptide, is
known as a competitive inhibitor of Myr59F-NTCP inter-
action. Notably, we found that both 27A and Myr47
significantly blocked Myr59F binding to NTCP, which is
revealed by decreased cell membrane staining compared with
DMSO control (Figure 4C). Myr47 inhibition has been
studied in NTCP-mediated TCA uptake assay using HepG2
cells expressing NTCP from multiple species and found
similarly high-affinity inhibition among human-NTCP, mouse-
NTCP, and treeshrew-NTCP but not monkey-NTCP (Figure
4D), owing to the sequence difference in the binding motif
residues 157−165, whereas 27A can block NTCP-mediated
TCA uptake in all species. We also tested both inhibitors
against another bile acid transporter protein ASBT, which is
localized in the terminal ileum, and found that both
compounds had no inhibition to ASBT’s function.
in HBV infection assays. Alisporivir inhibits HBV infection at
10 μM that is about 100-fold higher than 27A (Supplement
Figure 5), indicating that the mechanism of 27A inhibiting
HBV early infection is not related to cyclophilin inhibition.
Collectively, these results support a conclusion that 27A
inhibits HBV infection by disrupting the interaction between
NTCP and HBV Pre-S1 polypeptide, while stressing that 27A
may target a site on NTCP, which is distinct from the site
targeted by the HBV Pre-S1 polypeptide.
Biological Evaluation. We next evaluate the drug
potential of 27A. Given the knowledge that CsA has been
reported as an inhibitor for another liver-tropism bile acid
transportersorganic anion-transporting polypeptide (OATP)
family transporterscontributes to a multitude of transporter-
mediated drug−drug interactions in clinical situations.31−33
We therefore investigated the OATP inhibition activity of 27A
via uptake assay on OATP1B1 overexpressing HepG2 cells
using its natural substrate 3H-estrone sulfate. CsA, as expected,
We then compared Alisporivir, a cyclophilin inhibitor, which
was previously reported to suppress HBV replication with 27A
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Figure 3. 27A inhibits HDV infection in vivo. (A) Experimental scheme of in vivo HDV infection of mice models. (B) Intraperitoneal injection
(I.P.) of 27A inhibits HDV infection in mice. hNTCP-Tg-C57B/L mice were challenged with HDV by intraperitoneal injection of 5 × 109 Geq of
HDV on day 9 after birth and sacrificed on day 15 after birth. 27A was administrated once (Group A, n = 4), twice (Group B, n = 4), or vehicle
(Group C, n = 5) to mice through I.P. before HDV inoculation. HDV RNA levels were assessed by quantitative polymerase chain reaction (qPCR)
of the RNA extracted from the liver of challenged mice; the levels of NTCP mRNA were also determined. The RNA copy numbers from 20 ng of
liver RNA are presented. The bars indicate the median of each group. (C) Oral intake of 27A inhibits HDV infection in the mice. 27A (n = 4) or
vehicle (n = 4) were administrated to adult NTCP-edited FVB mice for 8 days through gavage. The mice were challenged with HDV on the 2nd
day by intraperitoneal injection of 5 × 109 Geq of HDV with 50 mg/kg dexamethasone. HDV RNA level and edited NTCP mRNA level were
determined in the same way as in (B).
significantly inhibits OATP1B1-mediated uptake of estrone
sulfate (estimated IC50 = 0.75 μM). In contrast, 27A inhibits
OATP1B1 at a much lower level compared with CsA
(estimated IC50 = 2.27 μM) (Figure 5A), indicating that
27A has lost affinity to OATP1B1 of its original structure CsA.
These results support that 27A has better selectively on NTCP
over OATP1B1 compared with CsA.
and ionomycin in the presence or absence of CsA or 27A (2
μM) for 24 h, the cells were then lysed, and the luciferase
activity was measured. CsA dramatically decreased the
luciferase activity compared to the DMSO control. In contrast,
the luciferase activity in 27A-treated cells was about 25 times
higher than that in CsA-treated cells (Figure 5C). These
results showed that the immunosuppressive effect of 27A is
largely blunted compared to its original structure.
Recall that CsA is widely used as an immunosuppressive
agent in the clinic, for example, in patients undergoing organ
transplantation. Considering the impressive results as an
antiviral agent, its potential development as an antiviral drug
would be aided by a lack of immunosuppressive activity. We
therefore investigated whether 27A retains any immunosup-
pressive activity. We separated primary spleen T cells from
adult C57B/L mouse spleens and stained them with CFSEa
fluorescent dye that diffuses equally into daughter cells,
enabling tracing of the proliferation of T cells.34 After
stimulating with anti-CD3 antibody in the presence or absence
of 27A or controls (CsA, DMSO) for 2 days, the cells were
then analyzed via FACS. The anti-CD3-stimulated primary
spleen T cells exhibited proliferation, which, as expected, was
totally abolished in the presence of CsA. In contrast, we found
that neither 27A nor DMSO abolished the proliferation of the
T cells, indicating that 27A has apparently lost the
immunosuppressive activity of its parent compound CsA
(Figure 5B).
CONCLUSIONS
■
In conclusion, we have completed the synthesis of cyclosporine
A analogues 1−53, all of which were screened using anti-HBV
infection assays with HepG2-NTCP cells. We ultimately
identified a highly potent and specific NTCP inhibitor, 27A,
which has a (3-(benzyloxy)phenyl)(methyl)sulfane group at
the C-5 position of the five-membered ring that contributes to
its low nanomolar potency. Virological studies indicated that
27A exerts pan-genomic HBV inhibition activity and HDV
inhibition activity in vitro. Supporting and extending these
findings, a series of in vivo studies showed that 27A can be used
as a prophylaxis agent for HDV infection and highlighted a
favorable pharmacokinetics profile and efficacy via oral dosage.
Subsequently, biochemical experiments showed that the
antiviral mechanism of 27A may be due to the inhibition of
the binding between HBV Pre-S1 peptide and NTCP, while
revealing that this mode of action does not rely on alteration of
the normal membrane localization of NTCP. In addition, 27A
is superior to CsA not only with better selectivity on NTCP
over OATP1B1 but also with the lack of immunosuppressive
effect, on the aspect of being an antiviral agent. Thus, our
We further verified this loss of immunosuppressive activity
by investigating the impacts of 27A on the calcineurin/NF-AT
pathway in Jurkat cells using a previously described pGL3-
NFAT-luc reporter assay.35 After stimulating cells with PMA
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3
Figure 4. Mechanism studies of 27A. (A) IC50 determination of 27A on inhibiting NTCP uptake of H-taurocholic acid (3H-TCA). (B) NTCP
(green) and cell nucleus (blue) staining using anti-NTCP antibody and DAPI, respectively. HepG2-NTCP cells were stained after 2 h treatment of
DMSO, 27A, or taurolithocholic acid (TLCA). The compounds were used at 2 μM. (C) Myr59F (green) staining on HepG2-NTCP cells in the
presence of DMSO, 27A (1 μM), or Myr47 (200 nM). The fluorescence intensity analysis is shown in Supplement Figure 3. (D) NTCP uptake
inhibition assay. 27A (2 μM) inhibits NTCP from multiple species (hNTCP: human-NTCP; mNTCP: mouse-NTCP; mkNTCP: monkey-NTCP;
3
tsNTCP: treeshrew-NTCP; hASBT: human-ASBT) uptake of H-TCA on transfected HepG2 cells, compared with tauroursodeoxycholic acid
(TUDCA, 20 μM) and Myr47 (200 nM).
plate reader (Beijing Pulang New Tech, Inc.). IC50 values (mean
standard deviation calculated from six or more measuring points in
three duplicate repeats) were determined using nonlinear regression
analysis by GraphPad software.
results collectively support that a small molecule that
specifically targets NTCP holds great promise for development
as an anti-HBV/HDV drug candidate and as a chemical tool to
study NTCP functions and the precise steps of the early phase
of HBV/HDV infection.
HBV Nucleic Acid Extraction, Southern and Northern
Blotting Assays. HBV cccDNA from HBV-infected cells was
extracted according to a modified Hirt method, as previously
described.5 Briefly, the infected cells from one well of six-well plates
were lysed in Hirt lysis buffer (10 mM Tris-HCl, 10 mM
ethylenediamine tetraacetic acid (EDTA), 0.6% sodium dodecyl
sulfate (SDS), pH 7.4) for 30 min at room temperature. After adding
5 M NaCl, the cell lysate was vigorously mixed and incubated at 4 °C
overnight. After centrifugation at 10 000 rpm for 30 min at 4 °C, the
supernatant was extracted twice with saturated Tris-phenol (pH 8.0)
and once with phenol-chloroform. The extracted DNA was
precipitated with equal volumes of isopropanol at −20 °C overnight.
The DNA pellet was washed with 70% ethanol and dissolved in TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to be analyzed.
HBV RNA from HBV-infected cells was extracted by TRIzol
reagent (Thermo Fisher) according to the manufacturer’s guide. The
infected cells from one well of a 24-well plate were lysed in TRIzol
reagent. After adding 1/5 volume of chloroform, the cell lysate was
mixed thoroughly and incubated for 5 min at room temperature. After
centrifugation at 12 000 rpm and 4 °C for 15 min, the supernatant
was transferred into a new tube, mixed with an equal volume of
EXPERIMENTAL SECTION
■
Ethics Statement. All animals were housed in the animal facility
of the National Institute of Biological Sciences (NIBS), Beijing.
Animal experiments were conducted following the National Guide-
lines for Housing and Care of Laboratory Animals and performed in
accordance with NIBS institutional regulations after approved by the
institution’s Institutional Animal Care and Use Committee (IACUC).
The protocol number is NIBS-0012.
IC50 Determination Using In Vitro HBV Infection System.
HepG2-NTCP cells were seeded in a 96-well plate (Corning) for 4−6
h before the medium was changed to primary maintenance medium
(PMM, formula previously reported5) for 20−24 h. The cells were
inoculated with HBV virus-containing medium in the presence of
testing compounds at different concentrations for 4 h at room
temperature and 37 °C afterward for 24 h. The inoculum medium was
then changed to PMM with 2% fetal bovine serum (Gibco) for every
2 days till day 5 post infection. The titer of HBeAg secreted in the
inoculum was tested by an ELISA kit (Wantai, Inc.) and read by a
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Figure 5. Biological evaluation of 27A. (A) OATP1B1 uptake inhibition assay. 27A inhibits OATP1B1 uptake of 3H-estrone sulfate on transfected
HepG2 cells, compared with CsA. (B, C) Immunosuppressive effect of 27A: (B) FACS analysis of mouse primary spleen T cells stained by CSFE.
The cells were stimulated by anti-CD3 antibody in the presence of indicated compounds for 48 h before analysis. Pos. control: cells stimulated by
antibody and no other treatment. (C) NF-AT luciferase assay in Jurkat T cells stimulated by PMA (10 ng/mL) and ionomycin (500 nM) in the
presence of indicated compounds for 48 h before analysis. The concentration of the compounds used in this assay is 2 μM.
isopropanol, and incubated for 5 min before centrifuged at 12 000
rpm and 4 °C for 10 min to get an RNA pellet. The pellet was then
washed with 70% ethanol and dissolved in DEPC-treated water.
For detection of cccDNA by Southern blot, the extracted HBV
cccDNA samples were subjected to 1.2% agarose gel electrophoresis.
For detection of RNA by Northern blot, the extracted RNA samples
were subjected to 1.0% agarose gel supplemented with MOPs buffer
and formaldehyde.
quenched by adding 300 μL of methanol. The samples were
vigorously vortexed for 1 min and then placed at −20 °C for 1 h.
The samples were then centrifuged at 3500 rpm for 15 min, and the
supernatant was collected for liquid chromatography−mass spec-
trometry (LC−MS) analysis.
HDV Production, Mouse Viral Inoculation, and Quantifica-
tion of HDV RNA Assays. HDV was produced as previously
described via transfection of two plasmids in Huh-7 cells.5 The mice
were inoculated with purified HDV by intraperitoneal injection.
Mouse liver samples were homogenized in liquid nitrogen
immediately after collection, and then lysed by TRIzol reagent
(Thermo Fisher) and RNA was extracted as described earlier. The
total RNA was reverse-transcribed into cDNA with PrimeScriptTM
RT-PCR Kit (Takara), and cDNA from 20 ng of RNA was used for
qPCR assay.
The blotting procedures for Southern blot and Northern blot are
identical. After electrophoresis, the nuclei acid stayed in the gel was
transferred onto Amersham Hybond-N+ membrane (GE Healthcare).
The Hybond-N+ membrane was cross-linked in a UV cross-linker
chamber with a UV energy dosage of 1200 mJ and followed by
probing with DiG-labeled HBV genotype D (accession number:
U95551.1) linear full-length genomic DNA that was prepared by a
DiG High Prime DNA Labeling and Detection Kit (Roche).
Hybridization was performed in PerfectHyb plus hybridization buffer
(Sigma) with prehybridization for 1 h at 50 °C and overnight
hybridization at 50 °C, followed by two washes in two kinds of wash
buffers (0.5 × SSC, 0.1% SDS, room temperature and 0.1 × SSC,
0.1% SDS, 65 °C, respectively). Chemiluminescence excitation on the
membrane was conducted according to the manufacturer’s guide
(Roche). The membrane was blocked by a blocking buffer for 30 min
followed by incubation with Anti-DiG-AP antibody (Roche) for 30
min. The membrane was then treated by CSPD solution (Roche) for
5 min and exposed to Carestream X-OMAT BT Film (XBT-1,
Carestream). For Southern blot assay, 100 pg each of 3.2, 2.1, and 1.7
kb HBV DNA fragments prepared by PCR amplification of a plasmid
containing 1.0 copies of linear HBV genotype D genome was used as
a DNA marker.
Liver Microsome Stability Assay. Commercial human/mouse
liver microsome was diluted to a final concentration of 1 mg/mL in
fresh 0.05 M phosphate buffer (the buffer was prepared by dissolving
8.7 g of K2HPO4 in 1000 mL of H2O; pH was adjusted to 7.4 using
H3PO4). Testing compounds were added to the liver microsome at a
final concentration of 1 μM, mixed well, and the mixture was then
preincubated at 37 °C for 5 min. The enzyme activity was initiated by
adding 1 mM NADPH. A small volume of the mixture (20 μL) was
collected at different time points (0, 5, 15, 30, and 60 min) and
3H-TCA Uptake Assays. HepG2-NTCP cells were seeded in 96-
well plates for 4−6 h before the medium was changed to PMM for
20−24 h. The cells were then treated with test compounds for 2 h,
then the culture medium was changed to 100 μL of Na+ Ringer’s
buffer supplemented with 2 μM 3H-TCA (PerkinElmer). The uptake
procedure was conducted at 37 °C for 10 min before the buffer was
removed, and the cells were then washed by phosphate-buffered saline
(PBS) twice. The cells were then treated with 50 μL of 1% Triton-
X100 (Sigma) in PBS to release the isotype labeled TCA. The
supernatant was then transferred to a reading plate with 200 μL of
Ultima Gold XR LSC-cocktail (PerkinElmer), and the plate was then
read by a MicroBeta TriLux LSC Counter (PerkinElmer).
Immunosuppression Assays. Spleen T cells from a mature
C57B/L mouse were separated and cultured in complete RPMI-1640
medium (Thermo Fisher). The cells were primarily stained by CFSE
(Thermo Fisher) following manufacturer’s guide and then cultured in
96-well plates coated by anti-CD3 antibody for 72 h in the presence of
CsA, 27A, and DMSO control. Before testing, the cells were stained
with PE-anti-CD4 antibody (BD Biosciences). T cell proliferation was
determined by FACS (BD FACS Arial II). For NF-AT signaling
pathway assay, Jurkat cells were cultured in complete RPMI-1640
supplemented with 10% FBS (Gibco) and Glutamax (Thermo
Fisher). pGL3-NFAT-LUC plasmid was purchased from Addgene.
The plasmid was transfected into Jurkat cells by electroporation
conducted by a Neon Transfection system (Thermo Fisher) with
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parameters following manufacturer’s instructions (pulse voltage: 1350
V, pulse width: 10 ms, pulse number: 3). Twenty-four hours after
transfection, the cells were stimulated with 10 ng/mL PMA (Sigma)
and 500 nM ionomycin (Beyotime Biotechnology) with testing
compounds or control for 48 h. After stimulation, the cells were then
lysed by passive lysis buffer (Promega) and the luciferase activity in
the cell lysis was determined by a Centro LB960 plate reader
(Berthold Tech).
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-33-((2R,3R)-5-methoxy-3-methyltetra-
hydrofuran-2-yl)-1,4,7,10,12,15,19,25,28-nonamethyl-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (3). Compound 2 (50 mg,
0.042 mmol) was dissolved in MeOH (5 mL), p-TsOH (7 mg, 0.042
mmol) was added to the above solution at 0 °C, then the whole
system was stirred at r.t. overnight. The solvent was concentrated; the
residue was dissolved in ethyl acetate (5 mL); the organic layer was
washed with saturated aq. NaCl (3 mL) and was separated, dried and
concentrated; and the crude product was purified by prep. HPLC to
Chemistry. All solvents and reagents were used directly as
obtained commercially unless otherwise noted (the synthesis methods
of mercapto heterocyclic and aromatic intermediates are shown in the
Supporting Information). All air- and moisture-sensitive reactions
were carried out under an atmosphere of dry argon with heat-dried
glassware and standard syringe techniques. LC−mass spectrometry
1
afford 36 mg of a white solid. Yield: 72%. H NMR (CDCl3, 400
MHz) δ 8.33 (d, J = 9.6 Hz, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.62 (d, J
= 8.8 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H). 5.66 (dd, J = 4.4, 10.8 Hz,
1H), 5.31 (dd, J = 4.0, 11.2 Hz, 1H), 5.19−5.23 (m, 1H), 5.13−5.17
(m, 1H), 5.07−5.10 (m, 2H), 4.95−5.01 (m, 1H), 4.84−4.87 (m,
2H), 4.76−4.81 (m, 1H), 4.66 (d, J = 14.0 Hz, 1H), 4.39 (t, J = 7.2
Hz, 1H), 4.28 (t, J = 6.8 Hz, 1H), 4.13 (dd, J = 4.8, 9.6 Hz, 1H), 3.47
(s, 3H), 3.38 (s, 3H), 3.27 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.04 (s,
3H), 2.69 (s, 3H), 2.67 (s, 3H), 2.28−2.35 (m, 1H), 2.11−2.18 (m,
1H), 2.01−2.07 (m, 3H), 1.88−1.97 (m, 3H), 1.60−1.72 (m, 5H),
1.44−1.52 (m, 3H), 1.31−1.34 (m, 3H), 1.24−1.26 (m, 4H), 1.12−
1.16 (m, 3H), 0.96−0.98 (m, 6H), 0.92−0.95 (m, 6H), 0.90−0.91
(m, 6H), 0.85−0.88(m, 14H), 0.79−0.81 (m, 5H), 0.72−0.75 (m,
4H). 13C NMR (100 MHz, CDCl3) δ 173.6, 173.2, 173.1, 172.7,
171.5, 171.2, 170.8, 170.8, 170.4, 169.8, 168.8, 130.9, 128.8, 105.3,
81.7, 65.5, 60.4, 57.9, 57.0, 55.4, 54.9, 54.8, 54.7, 54.6, 54.2, 50.0,
48.5, 48.4, 48.0, 44.7, 40.7, 40.2, 39.2, 39.1, 37.2, 36.2, 34.3, 32.4,
31.5, 31.3, 31.1, 30.5, 30.0, 29.9, 29.7, 29.6, 25.9, 24.8, 24.8, 24.7,
24.5, 23.7, 23.7, 23.5, 23.4, 21.9, 21.4, 21.3, 21.2, 20.1, 19.0, 18.6,
17.7, 15.0, 13.7, 9.9. HRMS (ESI): m/z [M + H]+ calcd for
C61H110N11O13 1204.8285, found 1204.8281.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
3 3 - ( ( 2 R , 3 R ) - 3 - m e t h y l t e t r a h y d r o f u r a n - 2 - y l ) -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (4). Step 1:
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((1R,2R)-1,4-dihydroxy-
2-methylbutyl)-30-ethyl-6,9,18,24-tetraisobutyl-3,21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-un-
decaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone
(5)
1
(MS) system: Waters 2545 (XBridge C18)/3100 mass detector. H
NMR spectra were recorded on a Varian 400 MHz spectrometer
(Varian, Inc., Palo Alto, CA) at ambient temperature with
tetramethylsilane as an internal standard. 13C NMR spectra were
recorded on a Varian 100 MHz spectrometer (with complete proton
decoupling) at ambient temperature. Chemical shifts are reported in
ppm. Data for 1H NMR are reported as follows: chemical shift (δ) in
ppm, multiplicity (s = singlet, bs = broad singlet, d = doublet, t =
triplet, q = quartet, m = multiplet), coupling constants, and
integration. High-resolution mass spectra were obtained by Agilent
Technologies 6540UHDAccurate-Mass Q-TOF LC/MS (Santa Clara,
CA). The samples were analyzed by high-performance liquid
chromatography (HPLC)/MS on a Waters Auto Purification LC/
MS system (3100 mass detector, 2545 binary gradient module, 2767
sample manager, and 2998 photodiode array detector), which was
purchased from Waters (Milford, MA). The system was equipped
with a Waters C18 5 μm SunFire separation column (150 × 4.6
mm2), equilibrated with HPLC-grade water (solvent A) and HPLC-
grade acetonitrile (solvent B) with a flow rate of 0.3 mL/min. Thin-
layer chromatography was carried out using HS-GF 254 silica plates;
TLC spots were visualized under UV light; chromatographic
purification was carried out using silica gel (Yan Tai Mou Ping
Yuan Bo Jing Xi Silica Gel Plant). The purity of all final compounds
was confirmed to be >95% by HPLC.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R)-5-
hydroxy-3-methyltetrahydrofuran-2-yl)-6,9,18,24-tetraisobutyl-
3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (2). To a well-stirred
solution of cyclosporine A (0.12 g, 0.1 mmol) in CH2Cl2 (5 mL)
was bubbled a flow of O3 at −78 °C. After 5 min, the solvent turned
out blue, which indicated complete consumption of starting material,
0.3 mL of Me2S was added to quench the reaction; the system was
warmed to r.t. and stirred for another 2 h. The solvent was evaporated
to dryness under vacuum to give 0.12 g of a white solid, and the crude
product was used directly for the next step without further
Compound 2 (0.3 g, 0.24 mmol) was dissolved in 5 mL of MeOH.
NaBH4 (45 g, 1.22 mmol) was added slowly to the solution at 0 °C. It
was stirred at r.t for 16 h. Water was added to quench the reaction and
extracted with EtOAc (10 mL × 3). The organic layers were
combined and evaporated to dryness and purified by prep-HPLC to
1
give 200 mg of a white solid. Yield: 69.9%. H NMR (400 MHz,
CDCl3) δ 8.20 (d, J = 10.0 Hz, 1H), 7.85 (d, J = 6.8 Hz, 1H), 7.52 (d,
J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 5.69 (dd, J = 4.0, 11.6 Hz,
1H), 5.33 (d, J = 7.2 Hz, 1H), 5.29 (dd, J = 3.6, 11.6 Hz, 1H), 5.12−
5.16 (m, 1H), 5.03−5.10 (m, 2H), 4.94−5.00 (m, 1H), 4.79−4.92
(m, 2H), 4.70 (d, J = 14.0 Hz, 1H), 4.65 (t, J = 9.2 Hz, 1H), 4.41−
4.51 (m, 1H), 3.95 (t, J = 6.4 Hz, 1H), 3.61−3.67 (m, 1H), 3.53 (s,
3H), 3.45−3.51 (m, 1H), 3.39 (s, 3H), 3.28 (s, 3H), 3.15 (s, 3H),
3.14 (s, 3H), 2.68(s, 3H), 2.67 (s, 3H), 2.38−2.48 (m, 1H), 2.08−
2.20 (m, 4H), 1.95−2.04 (m, 2H), 1.77−1.87 (m, 3H), 1.56−1.66
(m, 5H), 1.40−1.48 (m, 3H), 1.31−1.34 (m, 4H), 1.25−1.27 (m,
3H), 1.01−1.06 (m, 7H), 0.97−1.01 (m, 6H), 0.92−0.96 (m, 11H),
0.88−0.91 (m, 4H), 0.84−0.87 (m, 11H), 0.76 (d, J = 6.8 Hz, 3H).
Mass (ESI): m/z calcd for C60H109N11O13 1192.60, found 1193.80 [M
+ H]+.
1
purification. H NMR (CDCl3, 400 MHz) δ 8.61 (d, J = 8.8 Hz,
1H), 8.27 (d, J = 10.0 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.33 (d, J =
7.6 Hz, 1H), 5.64−5.66 (m, 1H), 5.43−5.47 (m, 1H), 5.33−5.36 (m,
1H), 5.13−5.15 (m, 2H), 5.05−5.09 (m, 1H), 4.77−4.83 (m, 2H),
4.71−4.73 (m, 2H), 4.60−4.62 (m, 1H), 4.49−4.52(m, 1H), 4.14−
4.19 (m, 1H), 3.97−3.98 (m, 1H), 3.47 (s, 3H), 3.39 (s, 3H), 3.29 (s,
3H), 3.10 (s, 3H), 3.06 (s, 3H), 2.67 (s, 3H), 2.65 (s, 3H), 2.48−2.55
(m, 1H), 2.29−2.33 (m, 1H), 2.11−2.16 (m, 3H), 2.01−2.05 (m,
3H), 1.62−1.68 (m, 5H), 1.42−1.44 (m, 3H), 1.27−1.31 (m, 9H),
1.23−1.25 (m, 5H), 1.15−1.17 (m, 4H), 1.04−1.07 (m, 3H), 0.97−
0.99 (m, 8H), 0.88−0.89 (m, 6H), 0.81−0.83 (m, 8H), 0.71−0.74
(m, 4H), 0.63−0.65 (m, 4H). 13C NMR (CDCl3, 100 MHz) δ 175.3,
172.8, 171.9, 171.4, 170.8, 170.7, 170.1, 170.0, 168.7, 168.0, 167.5,
97.0, 81.4, 77.3, 77.0, 76.7, 61.9, 59.3, 56.8, 54.2, 53.7, 50.1, 48.6,
48.5, 48.1, 45.8, 43.5, 41.4, 39.6, 39.1, 37.9, 36.8, 36.2, 35.4, 31.3,
30.8, 30.4, 30.4, 30.1, 29.9, 29.7, 26.4, 26.1, 25.4, 25.0, 24.8, 24.7,
24.2, 23.6, 23.2, 23.0, 22.2, 22.0, 21.8, 20.8, 20.0, 19.4, 18.2, 17.5,
17.3, 17.1, 16.7, 9.6. HRMS (ESI): m/z [M + H]+ calcd for
C60H108N11O13 1190.8128, found 1190.8034.
Step 2: To a cooled mixture of compound 5 (0.1 g, 0.084 mmol)
and Et3N (13 mg, 18 μL) was added MsCl (11 mg, 7 μL) and stirred
at 0 °C to r.t. overnight. The reaction was quenched with water; the
organic layer was separated; the aqueous layer was extracted with
ethyl acetate (3 × 3 mL); the organic phase was combined, dried, and
filtered; and the solvent was evaporated under vacuum. The residue
was purified by prep. HPLC, and 50 mg of a white solid was obtained,
1
total yield: 50%. H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 9.2 Hz,
1H), 8.07 (d, J = 6.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.48 (d, J =
553
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
J. Med. Chem. 2021, 64, 543−565

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
7.6 Hz, 1H), 5.66 (d, J = 8.0 Hz, 1H), 5.32 (d, J = 10.4 Hz, 1H), 5.19
(d, J = 10.8 Hz, 1H), 5.06−5.13 (m, 2H), 5.02 (d, J = 8.8 Hz, 1H),
4.92−4.98 (m, 1H), 4.77−4.84 (m, 2H), 4.68 (d, J = 14.0 Hz, 1H),
4.38 (t, J = 6.4 Hz, 1H), 3.97 (d, J = 6.8 Hz, 1H), 3.67−3.73 (m, 3H),
3.46 (s, 3H), 3.38 (s, 3H), 3.23 (s, 3H), 3.16 (s, 3H), 3.06 (s, 3H),
2.67(s, 3H), 2.66 (s, 3H), 2.28−2.38 (m, 1H), 1.92−2.19 (m, 6H),
1.54−1.80 (m, 6H), 1.38−1.48 (m, 3H), 1.31 (d, J = 6.8 Hz, 4H),
1.24 (d, J = 6.4 Hz, 3H), 1.10−1.190 (m, 2H), 0.96−1.04 (m, 11H),
0.83−0.93 (m, 25H), 0.78 (d, J = 6.0 Hz, 3H), 0.68 (d, J = 6.0 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 173.61, 173.36, 173.17, 173.11,
171.36, 171.19, 170.85, 170.78, 170.68, 169.89, 169.37, 82.70, 67.08,
60.25, 57.86, 57.08, 54.84, 54.72, 54.18, 49.91, 48.54, 48.24, 47.86,
44.64, 40.61, 39.14, 37.29, 36.16, 34.94, 33.34, 32.63, 31.56, 31.22,
31.14, 29.94, 29.86, 29.60, 25.43, 24.75, 24.64, 24.50, 24.21, 23.76,
23.74, 23.71, 23.64, 23.43, 21.81, 21.21, 20.76, 20.38, 20.21, 19.22,
19.22, 18.50, 18.35, 17.64, 14.93, 9.84. Mass (ESI): m/z calcd for
C60H107N11O12 1173.81, found 1174.80 [M + H]+.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R)-3-methyl-5-(prop-1-en-2-yl)tetrahydrofuran-2-yl)-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (7). Step 1:
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R)-5-acetyl-3-
methyltetrahydrofuran-2-yl)-30-ethyl-6,9,18,24-tetraisobutyl-3,21-dii-
s o p r o p y l - 1 , 4 , 7 , 1 0 , 1 2 , 1 5 , 1 9 , 2 5 , 2 8 - n o n a m e t h y l -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (6)
65.54, 60.87, 57.86, 57.10, 54.89, 54.51, 54.33, 50.02, 48.58, 48.31,
47.94, 44.61, 40.53, 40.42, 39.21, 39.10, 37.16, 36.81, 36.32, 32.87,
31.57, 31.28, 31.26, 30.52, 29.93, 29.82, 29.61, 26.02, 24.79, 24.66,
24.63, 24.24, 23.71, 23.68, 23.47, 21.89, 21.36, 20.81, 20.16, 19.43,
19.16, 18.75, 18.70, 18.55, 18.16, 17.75, 14.97, 13.72, 9.84. Mass
(ESI): m/z calcd for C63H111N11O12 1213.84, found 1214.81 [M +
H]+.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-33-((2R,3R)-5-isopropyl-3-methyltetra-
hydrofuran-2-yl)-1,4,7,10,12,15,19,25,28-nonamethyl-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (8). 10% Pd/C (10 mg)
and 6 N HCl (0.5 mL) were added to a solution of compound 7 (70
mg, 0.058 mmol) in MeOH (5 mL). The resulting suspension was
stirred under an atmosphere of H2 at room temperature overnight,
and then Pd/C was filtered with celite. It was then concentrated and
purified by prep. HPLC to obtain 8 mg of a white solid, yield: 11%.
1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 9.6 Hz, 1H), 8.06 (d, J =
7.2 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 5.66
(dd, J = 4.0, 11.2 Hz, 1H), 5.35 (dd, J = 4.0, 11.2 Hz, 1H), 5.24 (d, J
= 11.2 Hz, 1H), 5.12−5.18 (m, 1H), 5.07 (t, J = 6.8 Hz, 1H), 4.91−
5.03 (m, 2H), 4.87−4.89 (m, 1H), 4.84 (t, J = 7.2 Hz, 1H), 4.71−
4.73 (m, 1H), 4.65 (d, J = 14.0 Hz, 1H), 4.40 (t, J = 7.2 Hz, 1H), 4.28
(t, J = 6.8 Hz, 1H), 4.02−4.08 (m, 1H), 3.47 (s, 3H), 3.36 (s, 3H),
3.22 (s, 3H), 3.16 (s, 3H), 3.04 (s, 3H), 2.68 (s, 3H), 2.66 (s, 3H),
2.25−2.34 (m, 1H), 1.96−2.18 (m, 8H), 1.65−1.76 (m, 3H), 1.62−
1.64 (m, 3H), 1.55−1.61 (m, 2H), 1.41−1.47 (m, 5H), 1.33 (d, J =
7.2 Hz, 4H), 1.26 (d, J = 6.8 Hz, 4H), 1.04 (d, J = 6.4 Hz, 3H), 0.97−
1.01 (m, 10H), 0.91−0.96 (m, 10H), 0.84−0.89 (m, 14H), 0.78 (d, J
= 6.8 Hz, 4H), 0.68 (d, J = 6.4 Hz, 3H). Mass (ESI): m/z calcd for
C63H113N11O12 1215.86, found 1217.1 [M + H]+.
Cyclosporin A (100 mg, 0.083 mmol) was dissolved in 1 mL of
acetone. Jones reagent (0.3 mL) was added slowly to the solution and
stirred at r.t for 16 h. MeOH was added to quench the reaction. The
solvent was evaporated to dryness and purified by prep-TLC (DCM/
1
MeOH = 15/1) to give 25 mg of a brown solid. Yield: 24.6%. H
(4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-Ethyl-
1 1 , 1 7 , 2 6 , 2 9 - t e t r a i s o b u t y l - 1 4 , 3 2 - d i i s o p r o p y l -
1 , 7 , 1 0 , 1 6 , 2 0 , 2 3 , 2 5 , 2 8 , 3 1 - n o n a m e t h y l -
3 , 6 , 9 , 1 2 , 1 5 , 1 8 , 2 1 , 2 4 , 2 7 , 3 0 , 3 3 - u n d e c a o x o -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-4-
methyltetrahydrofuran-2-yl acetate (9). Compound 2 (50 mg,
0.042 mmol) and acetic anhydride (0.3 mL) were dissolved in
anhydrous pyridine (3 mL), and the solution was allowed to stand at
r.t. for 12 h. The solvent was concentrated and purified by prep.
HPLC to obtain 38 mg of a white solid, yield: 76%. 1H NMR (CDCl3,
400 MHz) δ 8.49 (d, J = 9.6 Hz, 1H), 8.33 (d, J = 9.6 Hz, 1H), 8.02
(d, J = 7.2 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 5.62−5.66 (m, 1H),
5.41−5.45 (m, 1H), 5.25−5.29 (m, 2H), 5.13−5.15 (m, 1H), 4.89−
4.97 (m, 2H), 4.74−4.80 (m, 2H), 4.55−4.62 (m, 1H), 4.42−4.50
(m, 1H), 4.34−4.37 (m, 1H), 4.24−4.30 (m, 1H), 3.94−4.10 (m,
1H), 3.43 (s, 3H), 3.36 (s, 3H), 3.23 (s, 3H), 3.14 (s, 3H), 2.75 (s,
3H), 2.66 (s, 3H), 2.63 (s, 3H), 2.36−2.42 (m, 1H), 2.29−2.34 (m,
1H), 2.06−2.14 (m, 3H), 1.99 (s, 3H), 1.91−1.96 (m, 3H), 1.67−
1.75 (m, 3H), 1.52−1.58 (m, 5H), 1.41−1.46 (m, 3H), 1.30−1.33
(m, 3H), 1.22−1.25 (m, 3H), 1.16−1.19 (m, 4H), 1.13−1.15 (m,
3H), 0.99−1.02 (m, 5H), 0.94−0.96 (m, 5H), 0.91−0.93 (m, 6H),
0.85−0.88 (m, 7H), 0.76−0.79 (m, 6H), 0.67−0.71 (m, 4H), 0.57−
0.62 (m, 2H). Mass (ESI): m/z calcd for C62H109N11O14 1231.82,
found 1232.86 [M + H]+.
NMR (400 MHz, CDCl3) δ 8.24 (d, J = 10.0 Hz, 1H), 7.95 (d, J = 7.2
Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 5.66 (dd,
J = 4.0, 10.8 Hz, 1H), 5.28 (dd, J = 8.0, 10.8 Hz, 1H), 5.16−5.21 (m,
2H), 5.07−5.15 (m, 2H), 4.95−5.01 (m, 1H), 4.80−4.89 (m, 3H),
4.69 (d, J = 14.0 Hz, 1H), 4.37−4.44 (m, 1H), 4.21 (dd, J = 5.2, 8.8
Hz, 1H), 4.04−4.11 (m, 1H), 3.48 (s, 3H), 3.37 (s, 3H), 3.22 (s, 3H),
3.17 (s, 3H), 3.04 (s, 3H), 2.68 (s, 3H), 2.66 (s, 3H), 2.28−2.35 (m,
1H), 2.22−2.25 (m, 1H), 2.19 (s, 3H), 1.91−2.13 (m, 6H), 1.54−
1.77 (m, 6H), 1.39−1.48 (m, 2H), 1.29−1.34 (m, 4H), 1.22−1.26
(m, 5H), 0.97−1.02 (m, 10H), 0.91−0.94(m, 8H), 0.84−0.88 (m,
16H), 0.81 (d, J = 6.4 Hz, 4H), 0.69 (d, J = 6.4 Hz, 4H). Mass (ESI):
m/z calcd for C62H109N11O13 1216.62, found 1217.80 [M + H]+.
Step 2: To a cooled solution of Ph3P+-CH3Br− (71.2 mg, 0.2
mmol) in THF (3 mL) was added t-BuOK (18 mg, 0.16 mmol) in
THF (2 mL). The suspension was stirred at −78 °C for 10 min and
then warmed to 0 °C for 30 min. The obtained orange solution was
cooled to −78 °C again, and a solution of compound 5 (100 mg,
0.082 mmol) in THF (2 mL) was added. The obtained mixture was
stirred at 0 °C for 3 h. The reaction was quenched with H2O; the
organic layer was separated; the aqueous phase was extracted with
ethyl acetate with three times (3 × 5 mL); and the organic phase was
combined, dried, filtered, and evaporated under vacuo. The residue
was purified by prep. HPLC. A white solid (23 mg) was obtained,
yield: 23%. 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 9.6 Hz, 1H),
8.05 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.46 (d, J = 8.0 Hz,
1H), 5.67 (dd, J = 4.0, 11.2 Hz, 1H), 5.34 (dd, J = 4.0, 11.2 Hz, 1H),
5.22 (d, J = 11.2 Hz, 1H), 5.11−5.18 (m, 2H), 5.07 (t, J = 6.8 Hz,
1H), 4.93−5.02 (m, 2H), 4.89 (s, 1H), 4.85 (t, J = 7.2 Hz, 1H), 4.73
(s, 1H), 4.66 (d, J = 14.0 Hz, 1H), 4.40 (t, J = 7.2 Hz, 1H), 4.29 (t, J
= 6.8 Hz, 1H), 4.02−4.08 (m, 2H), 3.48 (s, 3H), 3.37 (s, 3H), 3.22
(s, 3H), 3.16 (s, 3H), 3.06 (s, 3H), 2.69 (s, 3H), 2.66 (s, 3H), 2.24−
2.33 (m, 1H), 1.95−2.16 (m, 8H), 1.65−1.75 (m, 3H), 1.64 (s, 3H),
1.54−1.60 (m, 2H), 1.40−1.47 (m, 3H), 1.34 (d, J = 7.2 Hz, 4H),
1.25 (d, J = 6.8 Hz, 4H), 1.05 (d, J = 6.4 Hz, 3H), 0.98−1.01 (m,
9H), 0.92−0.96 (m, 9H), 0.85−0.88 (m, 14H), 0.79 (d, J = 6.8 Hz,
4H), 0.69 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
173.61, 173.30, 172.89, 172.85, 171.59, 171.17, 170.82, 170.72,
170.28, 169.89, 169.14, 145.36, 130.89, 128.79, 109.77, 82.55, 81.16,
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-(p-tolylthio)tetrahydrofuran-2-yl)-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (10A). To the solution of
compound 9 (360.9 mg, 0.292 mmol) and p-toluenethiol (72.4 mg,
0.584 mmol) in 18 mL of acetonitrile were added N,O-bis-
(trimethylsilyl)acetamide (0.286 mL, 1.168 mmol) and trimethylsilyl
trifluoromethanesulfonate (0.159 mL, 0.876 mmol). The solution was
stirred at 50 °C under nitrogen for 90 min. The reaction mixture was
evaporated to dryness and redissolved with DCM (20 mL). It was
washed with sat. NaHCO3 solution and brine, dried with Na2SO4,
filtered, and evaporated to dryness. The brown residue was purified by
prep. HPLC to give 140 mg of compound 10A and 42.2 mg of
compound 10m (α,β-mixture, major:minor = 1:3.3) as a white solid.
1
Total yield: 48.1%. H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 9.6
554
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
J. Med. Chem. 2021, 64, 543−565

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H), 8.07 (d, J = 6.8 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.53 (d, J
= 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 5.67
(dd, J = 4.0, 11.2 Hz, 1H), 5.42 (dd, J = 3.6, 7.6 Hz, 1H), 5.33 (dd, J
= 4.0, 11.2 Hz, 1H), 5.22 (d, J = 1.2 Hz, 1H), 5.08−5.14 (m, 3H),
4.97−5.01 (m, 1H), 4.90−4.95 (m, 1H), 4.81−4.88 (m, 2H), 4.65 (d,
J = 13.6 Hz, 1H), 4.35−4.40 (m, 1H), 4.29 (dd, J = 6.4, 9.2 Hz, 1H),
3.46 (s, 3H), 3.39 (s, 3H), 3.22 (s, 3H), 3.18 (s, 3H), 3.07 (s, 3H),
2.71 (s, 3H), 2.66 (s, 3H), 2.39−2.47 (m, 1H), 2.31 (s, 3H), 2.22−
2.27 (m, 1H), 2.05−2.12 (m, 3H), 1.93−2.01 (m, 3H), 1.60−1.70
(m, 5H), 1.41−1.52 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.26 (d, J =
6.8 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 0.99−1.03 (m, 9H), 0.93−0.95
(m, 8H), 0.89−0.91 (m, 4H), 0.85−0.87 (m, 11H), 0.81 (d, J = 6.4
Hz, 4H), 0.77 (d, J = 6.8 Hz, 3H), 0.64 (d, J = 6.8 Hz, 3H). HRMS
(ESI): m/z [M + H]+ calcd for C67H114N11O12S 1296.8369, found
1296.8365.
1 , 7 , 1 0 , 1 6 , 2 0 , 2 3 , 2 5 , 2 8 , 3 1 - n o n a m e t h y l -
3 , 6 , 9 , 1 2 , 1 5 , 1 8 , 2 1 , 2 4 , 2 7 , 3 0 , 3 3 - u n d e c a o x o -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-4-
methyltetrahydrofuran-2-yl)oxy)propanoic Acid (13). To a well-
stirred solution of compound 12 (145 mg, 0.114 mmol) in CH3OH
(4 mL) was added 1 N NaOH (2 mL) at 0 °C. The mixture was
stirred at r.t. for 4.5 h. Then, it was evaporated under a reduced
pressure to give a yellow liquid. 1 N HCl was added until pH 1. Then,
CH2Cl2 (2 × 50 mL) was added to the above system. The organic
layers were combined and washed with saturated aqueous NaCl (50
mL), dried, and concentrated. The crude product was purified by
prep. HPLC to afford 20 mg of a white solid. Yield: 14.0%. 1H NMR
(CDCl3, 400 MHz): δ 8.28 (d, J = 6.8 Hz, 1H), 8.06 (d, J = 7.2 Hz,
1H), 7.62 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 5.68 (dd, J =
4.0, 11.2 Hz, 1H), 5.35 (dd, J = 3.6, 10.8 Hz, 1H), 5.16−5.09 (m,
4H), 5.01−4.97(m, 2H), 4.88 (t, J = 7.4 Hz, 1H), 4.80 (t, J = 8.8 Hz,
1H), 4.67 (d, J = 13.2 Hz, 1H), 4.41 (t, J = 7.2 Hz, 1H), 4.17 (dd, J =
4.8, 8.8 Hz, 1H), 3.98−3.93 (m, 2H), 3.79−3.74 (m, 1H), 3.50 (s,
3H), 3.41 (s, 3H), 3.25 (s, 3H), 3.18 (s, 3H), 3.07 (s, 3H), 2.81−2.74
(m, 2H), 2.69 (s, 3H), 2.68 (s, 3H), 2.38- 2.36 (m, 2H), 2.20−2.04
(m, 4H), 1.67−1.58 (m, 3 H), 1.53−1.49 (m, 2H), 1.32−1.30 (m,
4H), 1.28−1.25 (m, 7H), 1.15 (d, J = 6.8 Hz, 3H), 1.06−1.03 (m,
4H), 1.01−0.99 (m, 4H), 0.96−0.94 (m, 6H), 0.93−0.92 (m, 3H),
0.90−0.87 (m, 13H), 0.85−0.83 (m, 9H), 0.73 (d, J = 6.8 Hz, 3H).
HRMS (ESI): m/z [M + H]+ calcd for C63H112N11O15 1262.8339,
found 1262.8309.
Methyl 2-(((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-
5-ethyl-11,17,26,29-tetraisobutyl-14,32-diisopropyl-
1 , 7 , 1 0 , 1 6 , 2 0 , 2 3 , 2 5 , 2 8 , 3 1 - n o n a m e t h y l -
3 , 6 , 9 , 1 2 , 1 5 , 1 8 , 2 1 , 2 4 , 2 7 , 3 0 , 3 3 - u n d e c a o x o -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-4-
methyltetrahydrofuran-2-yl)oxy)acetate (11). Compound 10 (50
mg, 0.039 mmol), methyl-2-hydroxyacetate (7 mg, 0.078 mmol),
silver trifluoromethanesulfonate (10 mg, 0.078 mmol), and activated 3
Å molecular sieves (100 mg) were stirred in dry CH2Cl2 (2 mL) for 2
h under a nitrogen atmosphere in a three-neck flask (10 mL). N-
iodosuccinimide (17.6 mg, 0.078 mmol) and activated 3 Å molecular
sieves (100 mg) in dry CH3CN (1 mL) in another three-neck flask
(10 mL) were stirred at r.t. for 2 h. Then, the mixture in the second
flask was added to the first one, stirred at r.t. for another 1 h to
complete the reaction. The mixture was filtered and diluted with
CH2Cl2. The organic layer was washed with saturated aqueous
Na2S2O3, dried over Na2SO4, filtered, and concentrated. The residue
was purified by prep. HPLC to give 24 mg of a colorless oil, yield:
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5R)-3-methyl-5-(phenylthio)tetrahydrofuran-2-yl)-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (14B). The title com-
pound was obtained as a colorless oil using a method similar to
1
that described for compound 9 in 4.8% yield in one step. H NMR
1
(400 MHz, CDCl3) δ 8.44 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 6.8 Hz,
1H), 7.71 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.40−7.46
(m, 2H), 7.27−7.32 (m, 1H), 7.17−7.22 (m, 2H), 5.65−5.69 (m,
1H), 5.47−5.50 (m, 1H), 5.32−5.37 (m, 1H), 5.18−5.24 (m, 2H),
5.07−5.14 (m, 2H), 4.98−5.04 (m, 1H), 4.90−4.96 (m, 1H), 4.84−
4.88 (m, 1H), 4.62−4.69 (m, 1H), 4.35−4.43 (m, 1H), 4.26−4.33
(m, 1H), 4.06−4.11 (m, 1H), 3.45 (s, 3H), 3.39 (s, 3H), 3.22 (s, 3H),
3.18 (s, 3H), 3.07 (s, 3H), 2.71 (s, 3H), 2.66 (s, 3H), 2.41−2.48 (m,
1H), 2.26−2.34 (m, 1H), 2.04−2.16 (m, 3H), 1.97−2.03 (m, 3H),
1.60−1.69 (m, 5H), 1.40−1.52 (m, 3H), 1.31−1.35 (m, 6H), 1.25−
1.27 (m, 9H), 1.15−1.19 (m, 4H), 1.01−1.05 (m, 6H), 0.93−0.96
(m, 7H), 0.85−0.89 (m, 13H), 0.75−0.77 (m, 3H), 0.60−0.63 (m,
3H). HRMS (ESI): m/z [M + H]+ calcd for C66H112N11O12S
1282.8213, found 1282.8212.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((4-(trifluoromethyl)pyrimidin-2-yl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (15A).
The title compound was obtained as a white solid using a method
similar to that described for compound 9 in 1.8% yield in one step. 1H
NMR (400 MHz, CDCl3) δ 8.74 (d, J = 5.2 Hz, 1H), 8.40 (d, J = 10.0
Hz, 1H), 8.06 (d, J = 6.8 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.52 (d, J
= 8.0 Hz, 1H), 7.29 (d, J = 6.8 Hz, 1H), 5.68 (dd, J = 4.0, 11.2 Hz,
1H), 5.33−5.36 (m, 2H), 5.16−5.21 (m, 3H), 4.95−5.01 (m, 2H),
4.89−4.92 (m, 1H), 4.84−4.87 (m, 2H), 4.68 (d, J = 13.6 Hz, 1H),
4.39−4.44 (m, 1H), 4.32−4.34 (m, 1H), 3.41 (s, 3H), 3.38 (s, 3H),
3.25 (s, 3H), 3.19 (s, 3H), 3.07 (s, 3H), 2.69 (s, 3H), 2.68 (s, 3H),
2.47−2.54 (m, 1H), 2.28−2.32 (m, 1H), 2.12−2.17 (m, 3H), 2.02−
2.06 (m, 3H), 1.63−1.71 (m, 6H), 1.45−1.49 (m, 2H), 1.33−1.35
(m, 6H), 1.26−1.28 (m, 3H), 1.00−1.03 (m, 8H), 0.93−0.96 (m,
12H), 0.87−0.89 (m, 12H), 0.80−0.83 (m, 7H), 0.77 (d, J = 6.4 Hz,
3H). HRMS (ESI): m/z [M + H]+ calcd for C65H109F3N13O12S
1352.7991, found 1352.7955.
48.8%. H NMR (CDCl3, 400 MHz): δ 8.35 (d, J = 10.0 Hz, 1H),
8.04 (d, J = 6.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 7.6 Hz,
1H), 5.67 (dd, J = 4.0, 10.4 Hz, 1H), 5.32 (dd, J = 4.0, 11.2 Hz, 1H),
5.21 (d, J = 10.8 Hz, 1H), 5.15−5.07 (m, 4H), 5.02−4.96 (m, 2H),
4.87−4.82 (m, 2H), 4.67 (d, J = 13.6 Hz, 1H), 4.39 (t, J = 7.2 Hz,
1H), 4.22 (dd, J = 4.8, 9.6 Hz, 1H), 4.12 (s, 2H), 3.73 (s, 3H), 3.47
(s, 3H), 3.40 (s, 3H), 3.24 (s, 3H), 3.18 (s, 3H), 3.06 (s, 3H), 2.69 (s,
3H), 2.66 (s, 3H), 2.37−2.31 (m, 2H), 2.15−2.00 (m, 6H), 1.70−
1.60 (m, 5H), 1.50−1.40 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.27 (m,
3H), 1.21 (d, J = 6.8 Hz, 3H), 1.03−1.00 (m, 8H), 0.95−0.92 (m,
8H), 0.89−0.88 (m, 4H), 0.87−0.84 (m, 15H), 0.81 (d, J = 6.4 Hz,
4H), 0.70 (d, J = 6.8 Hz, 3H). HRMS (ESI): m/z [M + H]+ calcd for
C63H112N11O15 1262.8339, found 1262.8299.
Methyl 3-(((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-
5-Ethyl-11,17,26,29-tetraisobutyl-14,32-diisopropyl-
1 , 7 , 1 0 , 1 6 , 2 0 , 2 3 , 2 5 , 2 8 , 3 1 - n o n a m e t h y l -
3 , 6 , 9 , 1 2 , 1 5 , 1 8 , 2 1 , 2 4 , 2 7 , 3 0 , 3 3 - u n d e c a o x o -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-4-
methyltetrahydrofuran-2-yl)oxy)propanoate (12). The title com-
pound was obtained as a yellow oil using a method similar to that
1
described for compound 11 in 34.1% yield in one step. H NMR
(CDCl3, 400 MHz): δ 8.36 (d, J = 10.0 Hz, 1H), 8.05 (d, J = 6.8 Hz,
1H), 7.58 (d, J = 9.2 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 5.67 (dd, J =
4.4, 10.8 Hz, 1H), 5.32 (dd, J = 4.0, 11.6 Hz, 1H), 5.22 (d, J = 11.2
Hz, 1H), 5.15−5.07 (m, 3H), 5.01−4.95 (m, 1H), 4.91 (d, J = 4.8 Hz,
1H), 4.88−4.84 (m, 2H), 4.67 (d, J = 14.0 Hz, 1H), 4.39 (t, J = 7.2
Hz, 1H), 4.15 (dd, J = 4.8, 9.6 Hz, 1H), 3.92−3.86 (m, 1H), 3.68 (s,
3H), 3.49 (s, 3H), 3.40 (s, 3H), 3.23 (s, 3H), 3.18 (s, 3H), 3.06 (s,
3H), 2.79−2.77 (m, 2H), 2.70 (s, 3H), 2.67 (s, 3H), 2.56 (t, J = 7.2
Hz, 2H), 2.37−2.30 (m, 2H), 2.17−2.07 (m, 3H), 2.05−1.99 (m,
3H), 1.70−1.57 (m, 5H), 1.56−1.46 (m, 3H), 1.33 (d, J = 7.2 Hz,
3H), 1.26 (m, 3H), 1.14 (d, J = 6.8 Hz, 3H), 1.02−1.00 (m, 8H),
0.95−0.92 (m, 8H), 0.89−0.88 (m, 4H), 0.87 (m, 5H), 0.87−0.84
(m, 10H), 0.81 (d, J = 6.8 Hz, 4H), 0.73 (d, J = 6.4 Hz, 3H). HRMS
(ESI): m/z [M + H]+ calcd for C64H114N11O15 1276.8496, found
1276.8472.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-33-((2R,3R,5S)-5-((4-methoxyphenyl)-
thio)-3-methyltetrahydrofuran-2-yl)-1,4,7,10,12,15,19,25,28-nona-
methyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacon-
3-(((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-
1 1 , 1 7 , 2 6 , 2 9 - t e t r a i s o b u t y l - 1 4 , 3 2 - d i i s o p r o p y l -
555
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
J. Med. Chem. 2021, 64, 543−565

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
tan-2,5,8,11,14,17,20,23,26,29,32-undecaone (16A)
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tetraiso-
butyl-3,21-diisopropyl-33-((2R,3R,5R)-5-((4-methoxyphenyl)thio)-3-
methyltetrahydrofuran-2-yl)-1,4,7,10,12,15,19,25,28-nonamethyl-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (16B). The title com-
pounds 16A and 16B were obtained as a white solid using a method
similar to that described for compound 9 in 47.1 and 7.5% yields in
one step, respectively.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((4-((4-methylpiperazin-1-yl)methyl)-
phenyl)thio)tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (18A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 21.1% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 9.2 Hz, 1H),
8.05 (d, J = 6.4 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 7.6 Hz,
1H), 7.39 (m, 2H), 7.16−7.23 (m, 2H), 5.64−5.68 (m, 1H), 5.51−
5.53 (m, 1H), 5.30−5.34 (m, 1H), 5.10−5.23 (m, 4H), 4.96−5.02
(m, 1H), 4.81−4.92 (m, 3H), 4.65 (d, J = 13.6 Hz, 1H), 4.37−4.42
(m, 1H), 4.29−4.31 (m, 1H), 3.54 (s, 2H), 3.42 (s, 3H), 3.38 (s, 3H),
3.22 (s, 3H), 3.18 (s, 3H), 3.06 (s, 3H), 2.94−2.99 (m, 2H), 2.82−
2.92 (m, 6H), 2.77 (s, 3H), 2.70 (s, 3H), 2.67 (s, 3H), 2.41−2.49 (m,
1H), 2.27−2.34 (m, 1H), 2.09−2.18 (m, 3H), 1.92−2.06 (m, 3H),
1.57−1.74 (m, 6H), 1.45−1.55 (m, 2H), 1.33 (d, J = 6.8 Hz, 3H),
1.26 (d, J = 7.2 Hz, 3H), 1.17 (d, J = 6.4 Hz, 3H), 0.99−1.04 (m,
8H), 0.93−0.95 (m, 10H), 0.84−0.90 (m, 14H), 0.79−0.83 (m, 7H),
0.69 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.72,
173.14, 173.03, 172.94, 171.39, 171.13, 170.82, 170.73, 170.29,
169.91, 168.56, 129.58, 86.22, 81.77, 61.36, 59.68, 57.89, 57.00, 54.91,
54.57, 54.11, 53.70, 50.03, 49.48, 48.65, 48.31, 47.99, 44.65, 43.55,
43.54, 41.31, 40.92, 39.21, 39.04, 37.20, 36.25, 35.24, 32.59, 31.56,
31.26, 31.19, 29.98, 29.92, 29.71, 29.66, 29.57, 25.88, 24.79, 24.68,
24.60, 24.45, 24.02, 23.69, 23.62, 23.46, 23.41, 23.36, 22.75, 21.88,
21.72, 21.35, 21.29, 21.24, 20.22, 20.07, 18.89, 18.60, 18.57, 18.50,
17.66, 15.02, 9.82. HRMS (ESI): m/z [M + H]+ calcd for
C72H124N13O12S 1394.9213, found 1394.9305.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((4-morpholinophenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (19A).
The title compound was obtained as a white solid using a method
similar to that described for compound 9 in 28.9% yield in one step.
1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6 Hz, 1H), 8.03 (d, J =
6.8 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.38
(d, J = 8.8 Hz, 2H), 6.81−7.00 (m, 2H), 5.66 (dd, J = 4.4, 10.8 Hz,
1H), 5.34 (d, J = 4.0 Hz, 1H), 5.31 (d, J = 4.4 Hz, 1H), 5.21 (d, J =
8.8 Hz, 1H), 5.19 (d, J = 6.0 Hz, 1H), 5.06−5.14 (m, 2H), 4.98−5.02
(m, 1H), 4.81−4.96 (m, 3H), 4.66 (d, J = 13.6 Hz, 1H), 4.35−4.42
(m, 1H), 4.25 (dd, J = 6.4, 8.8 Hz, 1H), 3.80−3.92 (m, 4H), 3.45 (s,
3H), 3.39 (s, 3H), 3.22 (s, 3H), 3.18 (s, 3H), 3.14−3.17 (m, 4H),
3.06 (s, 3H), 2.70 (s, 3H), 2.66 (s, 3H), 2.36−2.43 (m, 1H), 2.28−
2.33 (m, 1H), 2.05−2.13 (m, 3H), 1.93−2.00 (m, 3H), 1.57−1.65
(m, 4H), 1.42−1.50 (m, 2H), 1.33 (d, J = 7.2 Hz, 4H), 1.25 (d, J =
6.8 Hz, 4H), 1.15 (d, J = 6.8 Hz, 3H), 0.97−1.02 (m, 8H), 0.92−0.96
(m, 9H), 0.87−0.90 (m, 8H), 0.85−0.86 (m, 8H), 0.81 (d, J = 6.4 Hz,
3H), 0.79 (d, J = 6.4 Hz, 3H), 0.66 (d, J = 6.4 Hz, 3H). HRMS (ESI):
m/z [M + H]+ calcd for C70H119ClN12O13S 1367.8740, found
1367.8741.
1
Compound 16A. H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 9.6
Hz, 1H), 8.05 (d, J = 6.8 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.49 (d, J
= 8.0 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 9.2 Hz, 2H), 5.65
(dd, J = 4.0, 10.8 Hz, 1H), 5.32 (dd, J = 4.0, 11.2 Hz, 1H), 5.27−5.31
(m, 1H), 5.19 (t, J = 9.2 Hz, 2H), 5.04−5.12 (m, 3H), 4.95−5.01 (m,
1H), 4.89−4.93 (m, 1H), 4.82−4.86 (m, 1H), 4.65 (d, J = 14.0 Hz,
1H), 4.34−4.41 (m, 1H), 4.26 (dd, J = 6.4, 9.2 Hz, 1H), 3.78 (s, 3H),
3.46 (s, 3H), 3.38 (s, 3H), 3.21 (s, 3H), 3.17 (s, 3H), 3.05 (s, 3H),
2.70 (s, 3H), 2.65 (s, 3H), 2.34−2.41 (m, 1H), 2.25−2.31 (m, 1H),
2.07−2.15 (m, 3H), 1.93−1.98 (m, 3H), 1.59−1.68 (m, 5H), 1.41−
1.47 (m, 3H), 1.32 (d, J = 6.8 Hz, 3H), 1.24 (d, J = 6.8 Hz, 3H), 1.14
(d, J = 6.8 Hz, 3H), 0.99−1.01 (m, 4H), 0.97−0.98 (m, 4H), 0.93−
0.95 (m, 10H), 0.89−0.90 (m, 6H), 0.85−0.86 (m, 8H), 0.81 (d, J =
6.4 Hz, 4H), 0.74 (d, J = 6.4 Hz, 3H), 0.59 (d, J = 6.8 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 173.64, 173.06, 172.86, 171.41, 171.32,
171.14, 170.86, 170.70, 170.24, 169.80, 168.62, 159.12, 134.67,
133.64(2C), 126.14, 114.39(5C), 87.71, 81.60, 59.96, 57.85, 57.01,
55.21, 54.91, 54.54, 54.20, 50.03, 48.66, 48.00, 44.64, 41.20, 40.67,
39.22, 39.01, 37.12, 36.28, 35.27, 32.59, 31.56, 31.24, 31.18, 29.88,
29.65, 25.95, 24.80, 24.67, 24.59, 24.43, 23.69, 23.62, 23.45, 23.29,
21.88, 21.39, 21.13, 20.10, 20.00, 18.81, 18.67, 18.59, 17.70, 15.06,
9.82. HRMS (ESI): m/z [M + H]+ calcd for C67H114N11O13S
1312.8318, found 1312.8287.
1
Compound 16B. H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 9.6
Hz, 1H), 7.99 (d, J = 6.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.41 (d, J
= 8.8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.8 Hz, 2H), 5.68
(dd, J = 4.0, 11.2 Hz, 1H), 5.31 (dd, J = 4.0, 11.2 Hz, 1H), 5.18−5.24
(m, 3H), 5.07−5.16 (m, 3H), 4.92−4.98 (m, 1H), 4.83−4.88 (m,
2H), 4.67 (d, J = 14.0 Hz, 1H), 4.40−4.48 (m, 1H), 4.03 (dd, J = 5.6,
8.0 Hz, 1H), 3.79 (s, 3H), 3.54 (s, 3H), 3.27 (s, 3H), 3.25 (s, 3H),
3.17 (s, 3H), 3.07 (s, 3H), 2.70 (s, 6H), 2.30−2.39 (m, 1H), 2.20−
2.27 (m, 1H), 2.10−2.16 (m, 3H), 1.93−2.01 (m, 3H), 1.78−1.90
(m, 4H), 1.58−1.70 (m, 4H), 1.40−1.49 (m, 3H), 1.34 (d, J = 7.2 Hz,
3H), 1.25 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 0.99 (d, J =
6.8 Hz, 3H), 0.95−0.97 (m, 5H), 0.92−0.95 (m, 13H), 0.87−0.89
(m, 8H), 0.82−0.85 (m, 7H), 0.74 (d, J = 6.8 Hz, 3H). HRMS (ESI):
m/z [M + H]+ calcd for C67H114N11O13S 1312.8318, found
1312.8285.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((4-
Butylphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (17A). The title
compound was obtained as a white solid using a method similar to
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((4-
(Benzyloxy)phenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (20A). The title
compound was obtained as a white solid using a method similar to
1
that described for compound 9 in 9.2% yield in one step. H NMR
(400 MHz, CDCl3) δ 8.41 (d, J = 9.6 Hz, 1H), 8.06 (d, J = 6.8 Hz,
1H), 7.65 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.35 (d, J =
8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 5.68 (dd, J = 4.0, 11.2 Hz, 1H),
5.44 (dd, J = 3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.2 Hz, 1H), 5.22
(d, J = 6.4 Hz, 1H), 5.19 (d, J = 5.2 Hz, 1H), 5.08−5.15 (m, 2H),
4.97−5.02 (m, 1H), 4.91−4.95 (m, 1H), 4.83−4.87 (m, 2H), 4.66 (d,
J = 13.6 Hz, 1H), 4.35−4.43 (m, 1H), 4.29 (dd, J = 6.4, 9.6 Hz, 1H),
3.46 (s, 3H), 3.38 (s, 3H), 3.21 (s, 3H), 3.18 (s, 3H), 3.06 (s, 3H),
2.71 (s, 3H), 2.67 (s, 3H), 2.56 (t, J = 8.0 Hz, 2H), 2.41−2.45 (m,
1H), 2.26−2.29 (m, 2H), 2.06−2.18 (m, 5H), 1.93−2.02 (m, 3H),
1.55−1.65 (m, 7H), 1.34 (d, J = 6.8 Hz, 4H), 1.26 (d, J = 6.8 Hz,
4H), 1.16 (d, J = 6.8 Hz, 3H), 0.99−1.03 (m, 9H), 0.92−0.95 (m,
10H), 0.89−0.90 (m, 6H), 0.85−0.87 (m, 10H), 0.82 (d, J = 6.4 Hz,
4H), 0.78 (d, J = 6.4 Hz, 3H), 0.66 (d, J = 6.4 Hz, 3H). HRMS (ESI):
m/z [M + H]+calcd for C70H120N11O12S 1338.8839, found
1338.8806.
1
that described for compound 9 in 14.8% yield in one step. H NMR
(400 MHz, CDCl3): δ 8.38 (d, J = 9.2 Hz, 1H), 8.03 (d, J = 6.8 Hz,
1H), 7.65 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.36−7.41
(m, 7H), 6.92 (d, J = 8.8 Hz, 2H), 5.66 (dd, J = 3.6, 10.8 Hz, 1H),
5.30−5.36 (m, 2H), 5.21 (d, J = 8.8 Hz, 1H), 5.19 (d, J = 7.2 Hz,
1H), 5.05−5.13 (m, 3H), 5.04 (s, 2H), 4.99 (t, J = 7.2 Hz, 1H), 4.92
(t, J = 8.0 Hz, 1H), 4.85 (t, J = 7.2 Hz, 1H), 4.66 (d, J = 13.2 Hz, 1H),
4.36−4.42 (m, 1H), 4.27 (dd, J = 6.4, 8.8 Hz, 1H), 3.47 (s, 3H), 3.38
(s, 3H), 3.21 (s, 3H), 3.17 (s, 3H), 3.06 (s, 3H), 2.70 (s, 3H), 2.66 (s,
3H), 2.36−2.43 (m, 1H), 2.26−2.32 (m, 1H), 2.07−2.16 (m, 3H),
1.94−2.01 (m, 3H), 1.58−1.71 (m, 5H), 1.42−1.50 (m, 3H), 1.33 (d,
J = 7.2 Hz, 3H), 1.25−1.28 (m, 3H), 1.14 (d, J = 6.4 Hz, 3H), 1.01
(d, J = 6.4 Hz, 3H), 0.93−0.97 (m, 13H), 0.89−0.90 (m, 4H), 0.84−
556
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
J. Med. Chem. 2021, 64, 543−565

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
0.87 (m, 12H), 0.81 (d, J = 6.0 Hz, 4H), 0.77 (d, J = 6.4 Hz, 3H),
0.63 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/z [M + H]+ calcd for
C73H118N11O13S 1388.8631, found 1388.8594.
3H), 3.37 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s, 3H), 2.69 (s,
3H), 2.65 (s, 3H), 2.37−2.45 (m, 1H), 2.26−2.34 (m, 1H), 2.08−
2.16 (m, 3H), 1.94−2.04 (m, 3H), 1.53−1.70 (m, 6H), 1.44−1.51
(m, 2H), 1.40 (t, J = 6.8 Hz, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J
= 6.8 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H), 0.97−1.01 (m, 8H), 0.93−
0.95 (m, 9H), 0.89−0.91 (m, 4H), 0.84−0.88 (m, 11H), 0.81 (d, J =
6.4 Hz, 4H), 0.78 (d, J = 6.8 Hz, 3H), 0.61 (d, J = 6.4 Hz, 3H).
HRMS (ESI): m/z [M + H]+calcd for C68H116N11O13S 1326.8475,
found 1326.8461.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-propoxyphenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (24A).
The title compound was obtained as a white solid using a method
similar to that described for compound 9 in 21.5% yield in one step.
1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 9.6 Hz, 1H), 8.03 (d, J =
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-hydroxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (21A). The title
compound was obtained as a brown solid using a method similar to
1
that described for compound 9 in 2.3% yield in one step. H NMR
(400 MHz, CDCl3) δ 8.97 (brs, 1H), 8.44 (d, J = 9.2 Hz, 1H), 8.12
(d, J = 7.2 Hz, 1H), 7.74 (d, J = 9.2 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H),
7.29 (t, J = 2.0 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.74 (d, J = 8.0 Hz,
1H), 6.67−6.70 (m, 1H), 5.64 (dd, J = 4.0, 11.2 Hz, 1H), 5.58 (dd, J
= 3.6, 8.0 Hz, 1H), 5.34−5.38 (m, 3H), 5.24 (dd, J = 6.8, 9.2 Hz, 1H),
5.18 (t, J = 6.8 Hz, 1H), 4.99−5.05 (m, 2H), 4.86−4.91 (m, 2H),
4.65 (d, J = 13.2 Hz, 1H), 4.38−4.45 (m, 1H), 4.28 (dd, J = 8.0, 9.6
Hz, 1H), 3.44 (s, 3H), 3.43 (s, 3H), 3.24 (s, 3H), 3.18 (s, 3H), 3.09
(s, 3H), 2.73 (s, 3H), 2.69 (s, 3H), 2.32−2.42 (m, 1H), 2.14−2.21
(m, 2H), 1.98−2.11 (m, 3H), 1.88−1.97 (m, 2H), 1.62−1.70 (m,
5H), 1.41−1.49 (m, 3H), 1.31 (d, J = 7.2 Hz, 3H), 1.27 (d, J = 7.2
Hz, 3H), 1.26 (d, J = 4.8 Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H), 1.00−
1.04 (m, 6H), 0.91−0.95 (m, 14H), 0.86−0.89 (m, 12H), 0.84 (d, J =
2.8 Hz, 4H), 0.76 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/z [M + H]+
calcd for C66H112N11O13S 1298.8162, found 1298.8151.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-33-((2R,3R,5S)-5-((3-methoxyphenyl)-
thio)-3-methyltetrahydrofuran-2-yl)-1,4,7,10,12,15,19,25,28-nona-
methyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacon-
tan-2,5,8,11,14,17,20,23,26,29,32-undecaone (22A). The title
compound was obtained as a pale yellow solid using a method
similar to that described for compound 9 in 28.3% yield in one step.
1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6 Hz, 1H), 8.04 (d, J =
6.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.21
(t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.96 (t, J = 2.0 Hz, 1H),
6.74 (dd, J = 2.4, 8.0 Hz, 1H), 5.67 (dd, J = 4.0, 10.8 Hz, 1H), 5.48
(dd, J = 3.6, 8.0 Hz, 1H), 5.32 (dd, J = 3.6, 11.2 Hz, 1H), 5.16−5.20
(m, 2H), 5.06−5.12 (m, 3H), 4.95−5.01 (m, 1H), 4.92 (t, J = 8.0 Hz,
1H), 4.82−4.87 (m, 1H), 4.65 (d, J = 14.0 Hz, 1H), 4.36−4.43 (m,
1H), 4.32 (dd, J = 6.0, 9.2 Hz, 1H), 3.78 (s, 3H), 3.45 (s, 3H), 3.37
(s, 3H), 3.21 (s, 3H), 3.17 (s, 3H), 3.05 (s, 3H), 2.69 (s, 3H), 2.65 (s,
3H), 2.37−2.44 (m, 1H), 2.24−2.33 (m, 1H), 2.07−2.15 (m, 3H),
1.92−2.01 (m, 3H), 1.58−1.70 (m, 5H), 1.43−1.50 (m, 3H), 1.32 (d,
J = 7.2 Hz, 3H), 1.25 (d, J = 5.6 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H),
0.99−1.01 (m, 8H), 0.93−0.94 (m, 9H), 0.88−0.90 (m, 4H), 0.84−
0.86 (m, 11H), 0.80 (d, J = 6.4 Hz, 4H), 0.76 (d, J = 6.8 Hz, 3H),
0.60 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.70,
173.18, 173.10, 173.07, 171.48, 171.14, 170.85, 170.72, 170.33,
169.86, 168.66, 159.48, 137.41, 131.79, 129.72, 122.60, 115.16,
113.08, 87.00, 82.09, 60.09, 57.87, 57.03, 55.29, 54.88, 54.53, 54.20,
50.02, 48.62, 48.28, 47.96, 44.60, 41.14, 40.79, 39.22, 39.09, 37.21,
36.30, 35.04, 32.61, 31.63, 31.28, 31.20, 29.93,, 29.86, 29.64, 25.92,
24.82, 24.69, 24.55, 24.34, 23.84, 23.74, 23.61, 23.48, 23.35, 21.89,
21.39, 21.08, 20.50, 20.24, 18.81, 18.66, 18.58, 17.68, 14.95, 9.87.
HRMS (ESI): m/z [M + H]+ calcd for C67H114N11O13S 1312.8318,
found 1312.8298.
6.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.20
(t, J = 8.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.90 (t, J = 1.6 Hz, 1H),
6.73 (dd, J = 1.6, 8.0 Hz, 1H), 5.65 (dd, J = 4.0, 10.8 Hz, 1H), 5.46
(dd, J = 3.2, 7.6 Hz, 1H), 5.32 (dd, J = 3.2, 10.8 Hz, 1H), 5.17−5.20
(m, 2H), 5.04−5.12 (m, 3H), 4.94−5.00 (m, 1H), 4.91 (t, J = 8.0 Hz,
1H), 4.83 (t, J = 7.2 Hz, 1H), 4.64 (d, J = 13.6 Hz, 1H), 4.35−4.42
(m, 1H), 4.30 (dd, J = 6.4, 9.2 Hz, 1H), 3.88 (t, J = 6.4 Hz, 2H), 3.44
(s, 3H), 3.36 (s, 3H), 3.21 (s, 3H), 3.16 (s, 3H), 3.05 (s, 3H), 2.68 (s,
3H), 2.64 (s, 3H), 2.37−2.44 (m, 1H), 2.25−2.33 (m, 1H), 2.06−
2.15 (m, 3H), 1.93−2.02 (m, 3H), 1.73−1.82 (m, 2H), 1.57−1.66
(m, 5H), 1.41−1.50 (m, 3H), 1.32 (d, J = 7.2 Hz, 3H), 1.24 (d, J =
6.0 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 0.97−1.02 (m, 12H), 0.92−
0.94 (m, 8H), 0.88−0.89 (m, 4H), 0.83−0.86 (m, 11H), 0.79 (d, J =
6.8 Hz, 4H), 0.77 (d, J = 6.8 Hz, 3H), 0.61 (d, J = 6.4 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 173.65, 173.13, 173.08, 173.00, 171.45,
171.13, 170.82, 170.70, 170.30, 169.77, 168.65, 158.91, 137.09,
129.77, 122.52, 116.24, 113.35, 86.76, 81.95, 69.41, 59.90, 57.82,
57.01, 54.88, 54.53, 54.18, 50.00, 48.63, 48.30, 47.95, 44.60, 41.19,
40.77, 39.21, 39.03, 37.19, 36.26, 35.11, 32.61, 31.59, 31.25, 31.16,
29.89, 29.87, 29.74, 29.62, 25.88, 24.80, 24.66, 24.58, 24.37, 23.80,
23.70, 23.55, 23.44, 23.34, 22.51, 21.87, 21.37, 21.10, 20.21, 20.16,
18.82, 18.63, 18.56, 17.68, 14.97, 10.47, 9.82. HRMS (ESI): m/z [M
+ H]+ calcd for C69H118N11O13S 1340.8631, found 1340.8603.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
(Cyclopropylmethoxy)phenyl)thio)-3-methyltetrahydrofuran-2-yl)-
30-ethyl-6, 9, 18, 24-tetraisobutyl-3, 21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (25A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 18.3% yield in
1
one step. H NMR (400 MHz, CDCl3): δ 8.36 (d, J = 9.2 Hz, 1H),
8.05 (d, J = 6.4 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.49 (d, J = 7.6 Hz,
1H), 7.20 (t, J = 8.0 Hz, 1H), 7.08−7.11 (m, 1H), 6.89 (s, 1H), 6.74
(dd, J = 0.8, 8.0 Hz, 1H), 5.65 (dd, J = 3.2, 10.4 Hz, 1H), 5.46 (dd, J
= 3.6, 7.6 Hz, 1H), 5.31 (dd, J = 3.2, 10.8 Hz, 1H), 5.16−5.19 (m,
2H), 5.05−5.12 (m, 2H), 4.93−4.99 (m, 2H), 4.89 (t, J = 8.4 Hz,
1H), 4.83 (t, J = 7.6 Hz, 1H), 4.64 (d, J = 14.0 Hz, 1H), 4.34−4.41
(m, 1H), 4.29 (dd, J = 6.4, 8.4 Hz, 1H), 3.76 (d, J = 6.8 Hz, 2H), 3.43
(s, 3H), 3.35 (s, 3H), 3.20 (s, 3H), 3.16 (s, 3H), 3.04 (s, 3H), 2.68 (s,
3H), 2.65 (s, 3H), 2.38−2.45 (m, 1H), 2.24−2.30 (m, 1H), 2.03−
2.17 (m, 4H), 1.91−2.01 (m, 2H), 1.53−1.67 (m, 5H), 1.40−1.48
(m, 3H), 1.31 (d, J = 7.2 Hz, 3H), 1.24 (d, J = 6.4 Hz, 3H), 1.15 (d, J
= 6.4 Hz, 3H), 0.96−1.00 (m, 8H), 0.92−0.93 (m, 8H), 0.87−0.89
(m, 4H), 0.83−0.86 (m, 11H), 0.80 (d, J = 6.4 Hz, 4H), 0.76 (d, J =
6.8 Hz, 4H), 0.67−0.68 (m, 1H), 0.60−0.62 (m, 4H), 0.32 (q, J = 4.8
Hz, 3H). HRMS (ESI): m/z [M + H]+calcd for C70H118N11O13S
1352.8631, found 1352.8602.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-((tetrahydro-2H-pyran-4-yl)-
m e t h o x y ) p h e n y l ) t h i o ) t e t r a h y d r o f u r a n - 2 - y l ) -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (26A). The title com-
pound was obtained as a light yellow solid using a method similar
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
Ethoxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (23A). The title
compound was obtained as a light yellow solid using a method similar
1
to that described for compound 9 in 17.1% yield in one step. H
NMR (400 MHz, CDCl3) δ 8.38 (d, J = 9.6 Hz, 1H), 8.04 (d, J = 6.8
Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.21 (t, J
= 8.0 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.91 (t, J = 1.6 Hz, 1H), 6.73
(dd, J = 1.6, 7.6 Hz, 1H), 5.66 (dd, J = 4.0, 10.8 Hz, 1H), 5.48 (dd, J
= 3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.2 Hz, 1H), 5.21 (d, J = 6.4
Hz, 1H), 5.18 (d, J = 4.4 Hz, 1H), 5.06−5.13 (m, 2H), 4.92−5.02 (m,
2H), 4.81−4.87 (m, 2H), 4.65 (d, J = 13.6 Hz, 1H), 4.36−4.43 (m,
1H), 4.31 (dd, J = 6.0, 9.2 Hz, 1H), 4.00 (q, J = 6.8 Hz, 2H), 3.45 (s,
557
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J. Med. Chem. 2021, 64, 543−565

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Article
1
to that described for compound 9 in 14.2% yield in one step. H
NMR (400 MHz, CDCl3) δ 8.40 (d, J = 9.6 Hz, 1H), 8.05 (d, J = 6.8
Hz, 1H), 7.65 (d, J = 9.2 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.22 (t, J
= 8.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.73 (dd, J =
1.6, 7.6 Hz, 1H), 5.66 (dd, J = 4.0, 11.2 Hz, 1H), 5.47 (dd, J = 3.6, 7.6
Hz, 1H), 5.33 (dd, J = 3.6, 11.2 Hz, 1H), 5.21 (d, J = 5.2 Hz, 1H),
5.18 (d, J = 7.2 Hz, 1H), 5.06−5.13 (m, 2H), 4.96−5.01 (m, 1H),
4.89−4.93 (m, 1H), 4.78−4.89 (m, 2H), 4.66 (d, J = 13.6 Hz, 1H),
4.37−4.45 (m, 1H), 4.30 (dd, J = 6.4, 9.2 Hz, 1H), 4.01 (dd, J = 3.2,
11.2 Hz, 3H), 3.78 (d, J = 6.8 Hz, 3H), 3.44 (s, 3H), 3.37 (s, 3H),
3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s, 3H), 2.69 (s, 3H), 2.65 (s, 3H),
2.38−2.46 (m, 1H), 2.26−2.33 (m, 1H), 2.02−2.16 (m, 6H), 1.93−
2.00 (m, 2H), 1.74−1.77 (m, 3H), 1.58−1.69 (m, 5H), 1.40−1.49
(m, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.25 (d, J = 6.8 Hz, 3H), 1.17 (d, J
= 6.8Hz, 3H), 0.98−1.01 (m, 8H), 0.90−0.95 (m, 10H), 0.84−0.88
(m, 14H), 0.81 (d, J = 6.4 Hz, 4H), 0.77 (d, J = 6.4 Hz, 3H), 0.61 (d,
J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.68, 173.20,
173.12, 173.00, 171.51, 171.14, 170.82, 170.74, 170.32, 169.80,
168.67, 158.79, 137.30, 129.88, 122.67, 116.21, 113.28, 86.72, 81.94,
72.49, 70.93, 67.62, 59.93, 57.85, 57.06, 54.91, 54.56, 54.20, 50.02,
48.59, 48.29, 47.96, 44.59, 41.25, 40.84, 39.26, 39.22, 39.12, 37.26,
36.31, 35.16, 35.08, 32.61, 31.65, 31.30, 31.21, 29.91, 29.90, 29.72,
29.69, 29.63, 25.91, 24.83, 24.69, 24.60, 24.38, 23.87, 23.73, 23.67,
23.47, 23.42, 21.92, 21.40, 21.09, 20.31, 20.22, 18.90, 18.63, 18.59,
17.70, 14.90, 9.83. HRMS (ESI): m/z [M + H]+ calcd for
C72H122N11O14S 1396.8893, found 1396.8875.
(m, 1H), 4.08 (dd, J = 6.0, 6.8 Hz, 1H), 3.56 (s, 3H), 3.24 (s, 6H),
3.15 (s, 3H), 3.02 (s, 3H), 2.67 (s, 6H), 2.28−2.36 (m, 1H), 2.18−
2.24 (m, 1H), 2.05−2.14 (m, 3H), 1.90−1.99 (m, 3H), 1.76−1.87
(m, 2H), 1.54−1.68 (m, 3H), 1.35−1.46 (m, 3H), 1.33 (d, J = 7.2 Hz,
3H), 1.23−1.25 (m, 6H), 1.02 (d, J = 6.4 Hz, 3H), 0.97 (d, J = 6.8
Hz, 3H), 0.91−0.95 (m, 14H), 0.82−0.87 (m, 15H), 0.80 (d, J = 6.4
Hz, 4H), 0.67 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
173.94, 173.94, 173.86, 173.67, 173.36, 171.75, 171.37, 171.05,
170.89, 170.81, 170.34, 169.62, 159.08, 137.52, 137.08, 129.66,
128.75, 128.22, 127.99, 127.73, 122.99, 116.32, 112.88, 85.70, 84.43,
70.18, 59.89, 58.01, 57.45, 55.09, 54.98, 54.53, 49.87, 49.06, 48.52,
48.16, 44.82, 40.87, 40.59, 39.41, 39.23, 37.25, 36.34, 34.20, 33.70,
31.73, 31.54, 31.27, 30.19, 30.02, 29.90, 29.80, 25.61, 25.08, 24.88,
24.77, 24.35, 24.05, 23.97, 23.93, 23.66, 22.06, 21.43, 20.77, 20.66,
20.11, 19.43, 18.69, 18.08, 15.40, 10.15. HRMS (ESI): m/z [M +
H]+calcd for C73H118N11O13S 1388.8631, found 1388.8613.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-(1-phenylethoxy)phenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (28A).
The title compound was obtained as a white solid using a method
similar to that described for compound 9 in 18.1% yield in one step.
1H NMR (400 MHz, CDCl3): δ 8.36 (d, J = 9.6 Hz, 1H), 8.05 (d, J =
6.4 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.28−
7.35 (m, 5H), 7.20−7.24 (m, 1H), 7.03−7.05 (m, 1H), 6.85−6.90
(m, 1H), 6.66−6.68 (m, 1H), 5.65 (d, J = 9.2 Hz, 1H), 5.41−5.44 (m,
1H), 5.29−5.32 (m, 1H), 5.25−5.26 (m, 1H), 5.19 (s, 1H), 5.16 (s,
1H), 5.02−5.12 (m, 2H), 4.93−4.99 (m, 2H), 4.89 (t, J = 8.4 Hz,
1H), 4.83 (t, J = 6.8 Hz, 1H), 4.63 (d, J = 14.0 Hz, 1H), 4.34−4.39
(m, 1H), 4.24−4.28 (m, 1H), 3.42 (s, 3H), 3.34 (s, 3H), 3.20 (s, 3H),
3.16 (s, 3H), 3.04 (s, 3H), 2.68 (s, 3H), 2.65 (s, 3H), 2.35−2.43 (m,
1H), 2.24−2.30 (m, 1H), 2.03−2.13 (m, 4H), 1.92−2.00 (m, 2H),
1.60−1.67 (m, 5H), 1.59 (d, J = 6.0 Hz, 3H), 1.39−1.52 (m, 3H),
1.31 (d, J = 7.2 Hz, 3H), 1.24 (d, J = 6.0 Hz, 3H), 1.12 (d, J = 6.4 Hz,
3H), 0.95−1.00 (m, 8H), 0.92−0.94 (m, 11H), 0.84−0.88 (m, 13H),
0.75−0.80 (m, 7H), 0.62 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/z [M
+ H]+ calcd for C74H120N11O13S 1402.8788, found 1402.8754.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-Tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-phenethoxyphenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (29A).
The title compound was obtained as a white solid using a method
similar to that described for compound 9 in 2.0% yield in one step. 1H
NMR (400 MHz, CDCl3): δ 8.38 (d, J = 9.6 Hz, 1H), 8.06 (d, J = 6.8
Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.29−
7.33 (m, 5H), 7.20−7.24 (m, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.92 (s,
1H), 6.73−6.75 (m, 1H), 5.67 (dd, J = 3.2, 10.8 Hz, 1H), 5.47 (dd, J
= 3.6, 7.6 Hz, 1H), 5.31−5.37 (m, 2H), 5.20−5.22 (m, 1H), 5.18 (d, J
= 3.6 Hz, 1H), 5.11−5.13 (m, 1H), 5.08 (t, J = 6.8 Hz, 2H), 4.96−
5.01 (m, 2H), 4.91 (t, J = 8.4 Hz, 1H), 4.85 (t, J = 6.8 Hz, 1H), 4.66
(d, J = 13.6 Hz, 1H), 4.39 (t, J = 6.8 Hz, 1H), 4.31 (dd, J = 6.4, 8.8
Hz, 1H), 4.16 (dt, J = 2.4, 7.2 Hz, 2H), 3.43 (s, 3H), 3.37 (s, 3H),
3.22 (s, 3H), 3.18 (s, 3H), 3.06 (s, 3H), 2.70 (s, 3H), 2.66 (s, 3H),
2.39−2.44 (m, 1H), 2.28−2.31 (m, 1H), 2.09−2.15 (m, 3H), 1.93−
2.00 (m, 3H), 1.57−1.63 (m, 5H), 1.44−1.51 (m, 3H), 1.33 (d, J =
7.2 Hz, 3H), 1.26 (d, J = 6.8 Hz, 3H), 1.17 (d, J = 6.8 Hz, 3H), 0.98−
1.02 (m, 8H), 0.93−0.95 (m, 8H), 0.89−0.91 (m, 5H), 0.85−0.88
(m, 11H), 0.81 (d, J = 6.4 Hz, 4H), 0.78 (d, J = 6.8 Hz, 3H), 0.61 (d,
J = 6.8 Hz, 3H). HRMS (ESI): m/z [M + H]+ calcd for
C74H120N11O13S 1402.8788, found 1402.8752.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
(Benzyloxy)phenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (27A)
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5R)-5-((3-
(Benzyloxy)phenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (27B). The title
compounds 27A and 27B were obtained as a white solid using a
method similar to that described for compound 9 in 17.1% and 2.05%
yields in one step, respectively.
1
Compound 27A. H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 9.6
Hz, 1H), 8.06 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.51 (d, J
= 8.4 Hz, 1H), 7.43−7.44 (m, 2H), 7.35−7.39 (m, 2H), 7.29−7.33
(m, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.01−
7.04 (m, 1H), 6.82 (dd, J = 2.4, 8.0 Hz, 1H), 5.66 (dd, J = 4.0, 11.2
Hz, 1H), 5.49 (dd, J = 3.6, 7.6 Hz, 1H), 5.33 (dd, J = 4.0, 11.2 Hz,
1H), 5.21 (d, J = 6.0 Hz, 1H), 5.19 (d, J = 8.0 Hz, 1H), 5.07−5.14
(m, 2H), 5.05 (s, 2H), 4.98−5.02 (m, 1H), 4.83−4.97 (m, 3H), 4.65
(d, J = 14.0 Hz, 1H), 4.37−4.44 (m, 1H), 4.32 (dd, J = 6.0, 8.8 Hz,
1H), 3.46 (s, 3H), 3.37 (s, 3H), 3.25 (s, 3H), 3.17 (s, 3H), 3.06 (s,
3H), 2.69 (s, 3H), 2.65 (s, 3H), 2.39−2.47 (m, 1H), 2.26−2.33 (m,
1H), 2.07−2.15 (m, 3H), 1.93−2.05 (m, 3H), 1.55−1.70 (m, 5H),
1.42−1.49 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J = 7.2 Hz,
3H), 1.17 (d, J = 6.8 Hz, 3H), 0.97−1.04 (m, 8H), 0.93−0.95 (m,
8H), 0.84−0.90 (m, 16H), 0.81 (d, J = 6.8 Hz, 4H), 0.79 (d, J = 6.4
Hz, 3H), 0.62 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
173.83, 173.29, 173.24, 173.20, 171.58, 171.31, 171.00, 170.88,
170.49, 169.98, 168.81, 158.75, 137.47, 137.03, 130.02, 128.64(2C),
128.04, 127.64(2C), 127.58, 123.10, 116.75, 113.78, 86.85, 82.15,
70.07, 59.88, 58.04, 57.19, 55.07, 54.73, 54.35, 50.18, 48.85, 48.49,
48.14, 44.80, 41.37, 40.90, 39.38, 39.17, 37.35, 36.41, 35.30, 32.83,
31.74, 31.42, 31.32, 30.05, 29.86, 29.80, 26.02, 24.97, 24.84, 24.77,
24.55, 23.98, 23.87, 23.67, 23.62, 23.51, 22.04, 21.54, 21.35, 20.35,
20.26, 19.03, 18.80, 18.74, 17.87, 15.23, 9.99. HRMS (ESI): m/z [M
+ H]+calcd for C73H118N11O13S 1388.8631, found 1388.8595.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-(pyridin-2-ylmethoxy)phenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (30A).
The title compound 30A (30 mg) and the mixture 30m (10 mg,
major: minor = 1:1) were obtained as white solid using a method
similar to that described for compound 9 in total 17.8% yield in one
Compound 27B. 1H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 10.0
Hz, 1H), 7.97 (d, J = 6.8 Hz, 1H), 7.31−7.45 (m, 7H), 7.15 (t, J = 8.0
Hz, 1H), 7.13 (t, J = 2.4 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.79 (dd, J
= 2.4, 8.4 Hz, 1H), 5.67 (dd, J = 4.0, 11.2 Hz, 1H), 5.40 (dd, J = 6.0,
9.2 Hz, 1H), 5.31 (dd, J = 4.0, 11.2 Hz, 1H), 5.27−5.29 (m, 2H), 5.20
(d, J = 11.2 Hz, 1H), 5.04−5.09 (m, 2H), 5.03 (s, 2H), 4.89−4.95
(m, 1H), 4.79−4.86 (m, 2H), 4.55 (d, J = 13.6 Hz, 1H), 4.40−4.47
1
step. H NMR (400 MHz, CDCl3) δ 8.77 (brs, 1H), 8.39 (d, J = 9.6
558
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
J. Med. Chem. 2021, 64, 543−565

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H), 8.06 (d, J = 6.8 Hz, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.64 (d,
J = 9.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 8.0 Hz, 1H),
7.18 (d, J = 7.6 Hz, 1H), 7.03 (s, 1H), 6.83 (d, J = 7.2 Hz, 1H), 5.64
(dd, J = 4.0, 10.8 Hz, 1H), 5.49 (dd, J = 3.2, 7.6 Hz, 1H), 5.29−5.36
(m, 4H), 5.20 (d, J = 9.2 Hz, 1H), 5.17 (d, J = 11.2 Hz, 1H), 5.11
(dd, J = 5.6, 10.0 Hz, 1H), 5.06 (t, J = 6.8 Hz, 1H), 4.98 (dd, J = 8.4,
14.8 Hz, 1H), 4.90 (t, J = 8.4 Hz, 1H), 4.80−4.85 (m, 1H), 4.65 (d, J
= 14.0 Hz, 1H), 4.36−4.43 (m, 1H), 4.32 (dd, J = 6.4, 9.2 Hz, 1H),
3.45 (s, 3H), 3.37 (s, 3H), 3.21 (s, 3H), 3.16 (s, 3H), 3.05 (s, 3H),
2.65 (s, 3H), 2.64 (s, 3H), 2.40−2.48 (m, 1H), 2.25−2.34 (m, 1H),
2.07−2.14 (m, 3H), 1.93−2.03 (m, 3H), 1.54−1.70 (m, 6H), 1.41−
1.48 (m, 2H), 1.31 (d, J = 7.2 Hz, 3H), 1.24 (d, J = 6.4 Hz, 3H), 1.16
(d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.96−0.98 (m, 4H),
0.92−0.95 (m, 9H), 0.88−0.89 (m, 4H), 0.83−0.86 (m, 12H), 0.80
(d, J = 6.4 Hz, 4H), 0.77 (d, J = 6.4 Hz, 3H), 0.62 (d, J = 6.4 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 173.66, 173.11, 173.05, 173.01,
171.41, 171.14, 170.82, 170.72, 170.31, 169.82, 168.62,
157.81,156.21,138.62, 137.75, 130.03, 123.15, 116.68, 112.99, 86.51,
81.90, 59.66, 57.86, 57.00, 54.89, 54.56, 54.15, 50.01, 48.67, 48.30,
47.96, 44.62, 41.20, 40.73, 39.19, 39.02, 37.18, 36.24, 35.16, 32.65,
31.56, 31.25, 31.17, 29.89, 29.87, 29.72, 29.70, 29.64, 29.63, 29.29,
25.86, 24.79, 24.66, 24.59, 24.36, 23.80, 23.70, 23.52, 23.45, 23.32,
21.86, 21.35, 21.20, 20.19, 20.06, 18.85, 18.62, 18.54, 17.68, 16.76,
15.03, 9.82. HRMS (ESI): m/z [M + H]+ calcd for C72H117N12O13S
1389.8584, found 1389.8567.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-(pyridin-3-ylmethoxy)phenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (31A).
The title compound 31A (major, 40 mg) and the mixture 31m (10
mg, major: minor = 1:1.5) were obtained as white solid using a
method similar to that described for compound 9 in total 23.1% yield
in one step. 1H NMR (400 MHz, CDCl3) δ 8.90 (brs, 2H), 8.40 (d, J
= 9.6 Hz, 1H), 8.21 (d, J = 4.0 Hz, 1H), 8.06 (d, J = 6.8 Hz, 1H), 7.80
(brs, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.27 (t, J
= 8.0 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 6.83 (d, J = 8.0
Hz, 1H), 5.63 (dd, J = 4.0, 11.2 Hz, 1H), 5.48 (dd, J = 3.6, 7.6 Hz,
1H), 5.33 (dd, J = 3.6, 11.2 Hz, 1H), 5.21 (d, J = 9.2 Hz, 2H), 5.17
(d, J = 11.2 Hz, 2H), 5.11 (dd, J = 5.6, 10.4 Hz, 1H), 5.06 (t, J = 6.8
Hz, 1H), 4.95−5.01 (m, 1H), 4.89 (t, J = 8.4 Hz, 1H), 4.80−4.86 (m,
1H), 4.66 (d, J = 13.6Hz, 1H), 4.41−4.49 (m, 1H), 4.33−4.40 (m,
2H), 3.48 (s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s,
3H), 2.65 (s, 3H), 2.64 (s, 3H), 2.39−2.47 (m, 1H), 2.27−2.35 (m,
1H), 2.09−2.15 (m, 3H), 1.90−2.04 (m, 3H), 1.55−1.71 (m, 5H),
1.38−1.49 (m, 3H), 1.32 (d, J = 7.2 Hz, 3H), 1.24 (d, J = 7.2 Hz,
3H), 1.18 (d, J = 6.4 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.93−0.95
(m, 10H), 0.90−0.92 (m, 5H), 0.88−0.89 (m, 4H), 0.83−0.87 (m,
9H), 0.79−0.82 (m, 5H), 0.76 (d, J = 6.4 Hz, 3H), 0.58 (d, J = 6.4
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.73, 173.20, 173.12,
173.08, 171.36, 171.15, 170.86, 170.74, 170.38, 169.99, 168.68,
157.89, 137.83, 130.02, 123.68, 116.18, 113.86, 86.75, 82.23, 66.81,
59.55, 57.96, 57.01, 54.93, 54.63, 54.18, 50.04, 48.73, 48.31, 48.00,
44.65, 41.10, 40.69, 39.21, 39.03, 37.19, 36.26, 35.12, 32.67, 31.57,
31.27, 31.18, 29.93, 29.87, 29.75, 29.66(2C), 25.83, 24.82(2C), 24.68,
24.56, 24.32, 23.81(2C), 23.72(2C), 23.56, 23.47, 23.38, 21.86, 21.37,
21.15, 20.24, 20.14, 18.91, 18.62, 18.55, 17.71, 15.08, 14.35, 9.86.
HRMS (ESI): m/z [M + H]+ calcd for C72H117N12O13S 1389.8584,
found 1389.8593.
J = 4.0, 11.2 Hz, 1H), 5.47 (dd, J = 3.6, 7.6 Hz, 1H), 5.31 (dd, J = 4.0,
11.6 Hz, 1H), 5.29 (s, 2H), 5.19−5.24 (m, 2H), 5.16 (d, J = 10.8 Hz,
1H), 5.10 (dd, J = 5.6, 10.4 Hz, 1H), 5.06 (t, J = 6.8 Hz, 1H), 4.94−
5.00 (m, 1H), 4.88 (t, J = 8.4 Hz, 1H), 4.83 (t, J = 7.2 Hz, 1H), 4.65
(d, J = 13.6 Hz, 1H), 4.31−4.39 (m, 2H), 3.46 (s, 3H), 3.37 (s, 3H),
3.22 (s, 3H), 3.16 (s, 3H), 3.06 (s, 3H), 2.65 (s, 3H), 2.64 (s, 3H),
2.39−2.46 (m, 1H), 2.26−2.35 (m, 1H), 2.09−2.16 (m, 3H), 1.92−
2.04 (m, 3H), 1.54−1.72 (m, 6H), 1.39−1.46 (m, 2H), 1.31 (d, J =
7.2 Hz, 3H), 1.24 (d, J = 6.8 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.01
(d, J = 6.4 Hz, 3H), 0.92−0.96 (m, 13H), 0.87−0.89 (m, 7H), 0.83−
0.86 (m, 9H), 0.80−0.82 (m, 4H), 0.76 (d, J = 6.4 Hz, 3H), 0.58 (d, J
= 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.72, 173.18,
173.09, 173.04, 171.30, 171.11, 170.83, 170.72, 170.37, 170.00,
168.69, 157.49, 145.50, 145.47, 145.44, 138.02, 130.07, 123.77,
116.05, 113.68, 86.60, 82.28, 67.53, 59.44, 57.92, 56.97, 54.89, 54.61,
54.12, 50.00, 48.70, 48.26, 47.95, 44.62, 41.07, 40.70, 40.68, 39.19,
39.02, 37.16, 36.23, 35.09, 32.64, 31.54, 31.25, 31.16, 29.90, 29.85,
29.74, 29.63, 25.78, 24.79, 24.66, 24.52, 24.32, 24.28, 23.81, 23.70,
23.58, 23.45, 23.38, 21.83, 21.33, 21.11, 20.26, 20.04, 18.92, 18.57,
17.67, 16.39, 15.03, 9.84. HRMS (ESI): m/z [M + H]+ calcd for
C72H117N12O13S 1389.8584, found 1389.8556.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-((1-methyl-6-oxo-1,6-dihydropyridin-
3 - y l ) m e t h o x y ) p h e n y l ) t h i o ) t e t r a h y d r o f u r a n - 2 - y l ) -
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone (33A). The title com-
pound was obtained as a white solid using a method similar to that
described for compound 9 in 12.7% yield in one step. 1H NMR (400
MHz, CDCl3) δ 8.41 (d, J = 9.2 Hz, 1H), 8.06 (d, J = 6.8 Hz, 1H),
7.67 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.38−7.44 (m,
1H), 7.23−7.25 (m, 1H), 7.16−7.17 (m, 1H), 6.95−6.97 (m, 1H),
6.83−6.87 (m, 1H), 6.76−6.80 (m, 1H), 6.44−6.49 (m, 1H), 5.65
(dd, J = 4.0, 11.2 Hz, 1H), 5.49 (dd, J = 3.2, 7.6 Hz, 1H), 5.34 (dd, J
= 4.0, 10.8 Hz, 1H), 5.22 (d, J = 9.2 Hz, 1H), 5.18 (d, J = 10.8 Hz,
1H), 5.05−5.14 (m, 2H), 4.92−5.02 (m, 4H), 4.81−4.91 (m, 2H),
4.66 (d, J = 13.6 Hz, 1H), 4.37−4.44 (m, 1H), 4.32 (dd, J = 6.4, 9.2
Hz, 1H), 3.64 (s, 3H), 3.46 (s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.17
(s, 3H), 3.07 (s, 3H), 2.68 (s, 3H), 2.66 (s, 3H), 2.40−2.48 (m, 1H),
2.28−2.34 (m, 1H), 2.09−2.17 (m, 3H), 1.95−2.05 (m, 3H), 1.60−
1.72 (m, 5H), 1.40−1.51 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J
= 6.8 Hz, 3H), 1.17 (d, J = 6.8Hz, 3H), 0.97−1.02 (m, 9H), 0.92−
0.95 (m, 10H), 0.85−0.89 (m, 13H), 0.81 (d, J = 6.8 Hz, 4H), 0.78
(d, J = 6.4 Hz, 3H), 0.62 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/z [M
+ H]+ calcd for C73H119N12O14S 1419.8689, found 1419.8671.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-(methylamino)phenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (34A).
The title compound was obtained as a light yellow solid using a
method similar to that described for compound 9 in 13.1% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6 Hz, 1H),
8.09 (d, J = 6.4 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 8.4 Hz,
1H), 7.15 (t, J = 7.6 Hz, 1H), 7.09 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H),
6.66 (d, J = 7.6 Hz, 1H), 5.64 (dd, J = 3.2, 10.8 Hz, 1H), 5.53 (dd, J =
2.8, 8.0 Hz, 1H), 5.34 (dd, J = 3.2, 10.8 Hz, 1H), 5.25 (d, J = 9.6 Hz,
1H), 5.15 (dd, J = 6.4, 9.2 Hz, 1H), 5.08−5.11 (m, 2H), 4.97−5.03
(m, 1H), 4.84−4.93 (m, 2H), 4.66 (d, J = 14.0 Hz, 1H), 4.40 (t, J =
7.2 Hz, 1H), 4.29−4.33 (m, 2H), 3.45 (s, 3H), 3.40 (s, 3H), 3.22 (s,
3H), 3.17 (s, 3H), 3.07 (s, 3H), 2.89 (s, 3H), 2.70 (s, 3H), 2.69 (s,
3H), 2.47−2.54 (m, 1H), 2.29−2.37 (m, 1H), 2.11−2.17 (m, 2H),
2.03−2.07 (m, 1H), 1.93−2.01 (m, 2H), 1.83−1.88 (m, 1H), 1.55−
1.70 (m, 5H), 1.40−1.50 (m, 3H), 1.31 (d, J = 7.2 Hz, 3H), 1.26 (d, J
= 6.8 Hz, 3H), 1.16 (d, J = 6.8 z, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.93−
0.98 (m, 14H), 0.87−0.91 (m, 11H), 0.77−0.85 (m, 11H), 0.68 (d, J
= 6.4 Hz, 3H). HRMS (ESI): m/z [M + H]+ calcd for
C67H115N12O12S 1311.8478, found 1311.8447.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-6,9,18,24-tet-
raisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl-
33-((2R,3R,5S)-3-methyl-5-((3-(pyridin-4-ylmethoxy)phenyl)thio)-
tetrahydrofuran-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacy-
clotritriacontan-2,5,8,11,14,17,20,23,26,29,32-undecaone (32A).
The title compound was obtained as a white solid using a method
similar to that described for compound 9 in 13.3% yield in one step.
1H NMR (400 MHz, CDCl3) δ 8.90 (brs, 2H), 8.40 (d, J = 9.6 Hz,
1H), 8.04 (d, J = 7.2 Hz, 1H), 7.83 (brs, 2H), 7.62 (d, J = 9.2 Hz,
1H), 7.51 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0
Hz, 1H), 7.02−7.04 (m, 1H), 6.82 (dd, J = 2.0, 8.0 Hz, 1H), 5.63 (dd,
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-(ethyl(methyl)amino)phenyl)thio)-3-methyltetrahydrofuran-
2 - y l ) - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
559
https://dx.doi.org/10.1021/acs.jmedchem.0c01484
J. Med. Chem. 2021, 64, 543−565

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (35A). The title compound was obtained as a light yellow solid
using a method similar to that described for compound 9 in 6.8%
yield in one step. 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 9.6 Hz,
1H), 8.07 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.53 (d, J =
8.0 Hz, 1H), 7.40−7.44 (m, 2H), 7.29−7.35 (m, 1H), 7.19−7.23 (m,
1H), 5.66 (dd, J = 4.0, 11.6 Hz, 1H), 5.55 (dd, J = 3.2, 7.6 Hz, 1H),
5.33 (dd, J = 4.0, 11.6 Hz, 1H), 5.23 (d, J = 9.2 Hz, 1H), 5.09−5.16
(m, 3H), 4.97−5.01 (m, 1H), 4.89−4.95 (m, 2H), 4.82−4.87 (m,
1H), 4.66 (d, J = 13.6 Hz, 1H), 4.37−4.43 (m, 1H), 4.34 (dd, J = 6.4,
9.2 Hz, 1H), 3.48 (q, J = 6.8 Hz, 2H), 3.43 (s, 3H), 3.38 (s, 3H), 3.22
(s, 3H), 3.18 (s, 3H), 3.09 (s, 3H), 3.06 (s, 3H), 2.69 (s, 3H), 2.67 (s,
3H), 2.43−2.50 (m, 1H), 2.26−2.35 (m, 1H), 2.11−2.17 (m, 3H),
1.93−2.03 (m, 3H), 1.59−1.71 (m, 5H), 1.41−1.51 (m, 3H), 1.32 (d,
J = 6.8 Hz, 3H), 1.26 (d, J = 7.2 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H),
1.15 (t, J = 6.8 Hz, 3H), 0.97−1.02 (m, 9H), 0.93−0.95 (m, 8H),
0.84−0.90 (m, 15H), 0.79−0.83 (m, 7H), 0.67 (d, J = 6.4 Hz, 3H).
HRMS (ESI): m/z [M + H]+ calcd for C69H119N12O12S 1339.8791,
found 1339.8862.
3H). 13C NMR (100 MHz, CDCl3) δ 173.67, 173.09(2C), 173.05,
171.37, 171.12, 170.87, 170.70, 170.29, 169.88, 168.66, 148.77,
148.58, 126.57, 124.67, 115.21, 111.47, 87.99, 82.12, 60.04, 57.87,
56.97, 55.91, 55.82, 54.89, 54.53, 54.19, 50.01, 48.68, 48.28, 48.00,
44.62, 41.01, 40.57, 39.19, 38.99, 37.09, 36.26, 35.06, 32.62, 31.51,
31.20, 31.15, 29.88, 29.86, 29.73, 29.62, 25.91, 24.79, 24.65, 24.52,
24.36, 23.67, 23.60, 23.49, 23.43, 23.28, 21.84, 21.35, 21.15, 20.32,
20.15, 18.71, 18.68, 18.54, 17.66, 15.04, 9.84. HRMS (ESI): m/z [M
+ H]+calcd for C68H115N11O14S 1342.8424, found 1342.8405.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3,5-
Dimethoxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (38A). The title
compound was obtained as a white solid using a method similar to
1
that described for compound 9 in 28.4% yield in one step. H NMR
(400 MHz, CDCl3) δ 8.37 (d, J = 10.0 Hz, 1H), 8.03 (d, J = 7.2 Hz,
1H), 7.57 (d, J = 9.2 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 6.62−6.65
(m, 2H), 6.31−6.32 (m, 1H), 5.65 (dd, J = 3.2, 10.8 Hz, 1H), 5.46
(dd, J = 2.4, 7.6 Hz, 1H), 5.33 (dd, J = 3.6, 10.8 Hz, 1H), 5.20 (d, J =
11.2 Hz, 1H), 5.05−5.12 (m, 3H), 4.93−5.01 (m, 2H), 4.81−4.88
(m, 2H), 4.66 (d, J = 13.6 Hz, 1H), 4.34−4.43 (m, 2H), 3.78 (s, 3H),
3.77 (s, 3H), 3.49 (s, 3H), 3.37 (s, 3H), 3.21 (s, 3H), 3.17 (s, 3H),
3.06 (s, 3H), 2.69 (s, 3H), 2.65 (s, 3H), 2.25−2.35 (m, 2H), 2.10−
2.20 (m, 3H), 1.92−2.00 (m, 3H), 1.54−1.67 (m, 5H), 1.39−1.47
(m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.22−1.26 (m, 6H), 0.97−1.01 (m,
8H), 0.91−0.95 (m, 10H), 0.83−0.89 (m, 14H), 0.80 (d, J = 6.4 Hz,
4H), 0.76 (d, J = 6.4 Hz, 3H), 0.58 (d, J = 6.8 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 173.72, 173.32, 173.26, 173.10, 171.47, 171.11,
170.87, 170.68, 170.38, 169.93, 168.74, 160.62(2C), 137.81, 108.33,
100.13, 87.75, 82.87, 60.52, 57.87, 56.99, 55.44, 55.39, 55.32, 54.82,
54.47, 54.23, 50.01, 48.62, 48.18, 47.96, 44.54, 40.85, 40.58, 39.21,
39.07, 37.20, 36.29, 34.68, 32.61, 31.59, 31.21, 31.13, 30.03, 29.91,
29.87, 29.59, 25.90, 24.81, 24.66, 24.44, 24.21, 23.73, 23.69, 23.65,
23.43, 23.30, 21.87, 21.38, 21.10, 21.01, 20.28, 18.71, 18.63, 18.52,
17.64, 14.85, 9.90. HRMS (ESI): m/z [M + H]+ calcd for
C68H116N11O14S 1342.8424, found 1342.8416.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
(Benzylamino)phenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-
e t h y l - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (36A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 49.3% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 9.6 Hz, 1H),
8.09 (d, J = 6.8 Hz, 1H), 7.65 (d, J = 9.2 Hz, 1H), 7.54 (d, J = 8.0 Hz,
1H), 7.35 (d, J = 7.2 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.6
Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.70 (d, J = 7.6 Hz,
1H), 6.39 (dd, J = 1.6, 8.0 Hz, 1H), 5.64 (dd, J = 4.0, 10.8 Hz, 1H),
5.55 (dd, J = 3.2, 7.6 Hz, 1H), 5.34 (dd, J = 3.6, 11.2 Hz, 1H), 5.24
(d, J = 9.2 Hz, 1H), 5.16 (dd, J = 6.4, 9.2 Hz, 1H), 5.09 (d, J = 11.2
Hz, 1H), 5.05 (d, J = 7.2 Hz, 1H), 4.97−5.01 (m, 1H), 4.92 (t, J = 8.0
Hz, 1H), 4.86 (t, J = 7.2 Hz, 1H), 4.65 (d, J = 13.6 Hz, 1H), 4.40 (d, J
= 6.4 Hz, 1H), 4.36 (d, J = 12.4 Hz, 2H), 4.28−4.32 (m, 2H), 3.45 (s,
3H), 3.40 (s, 3H), 3.22 (s, 3H), 3.16 (s, 3H), 3.07 (s, 3H), 2.66 (s,
3H), 2.53 (s, 3H), 2.28−2.37 (m, 1H), 2.08−2.17 (m, 2H), 1.92−
2.05 (m, 5H), 1.57−1.69 (m, 5H), 1.40−1.49 (m, 3H), 1.32 (d, J =
7.2 Hz, 3H), 1.25 (d, J = 6.8 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.01
(d, J = 6.4 Hz, 3H), 0.92−0.97 (m, 13H), 0.87−0.91 (m, 10H), 0.81−
0.86 (m, 13H), 0.70 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 173.77, 173.12, 173.08, 172.77, 171.36, 171.20, 170.82,
170.29, 170.25, 168.67, 137.41, 129.45, 128.47, 127.32, 127.03,
118.15, 114.25, 85.55, 80.82, 80.80, 59.64, 58.18, 57.16, 54.96, 54.58,
54.15, 50.02, 48.67, 48.24, 48.21, 48.00, 44.54, 41.69, 40.42, 39.20,
39.12, 37.26, 36.34, 35.61, 32.71, 31.91, 31.59, 31.30, 31.24, 30.06,
29.76, 29.68, 29.63, 29.60, 29.35, 29.31, 25.93, 24.84, 24.68, 24.61,
24.25, 23.74, 23.65, 23.50, 23.30, 22.68, 21.91, 21.47, 21.41, 20.40,
19.95, 18.90, 18.64, 18.59, 17.92, 14.96, 14.11, 9.86. HRMS (ESI): m/
z [M + H]+ calcd for C73H119N12O12S 1387.8791, found 1387.8770.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3,4-
Dimethoxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (37A). The title
compound was obtained as a white foam using a method similar to
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
Chloro-5-methoxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-
e t h y l - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (39A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 16.1% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6 Hz, 1H),
8.05 (d, J = 6.8 Hz, 1H), 7.59 (d, J = 9.2 Hz, 1H), 7.54 (d, J = 8.4 Hz,
1H), 7.03 (dd, J = 1.6, 3.2 Hz, 1H), 6.89 (dd, J = 1.2, 2.0 Hz, 1H),
6.74 (t, J = 2.0 Hz, 1H), 5.66 (dd, J = 4.0, 11.2 Hz, 1H), 5.49 (dd, J =
2.4, 7.6 Hz, 1H), 5.32 (dd, J = 4.0, 10.8 Hz, 1H), 5.21 (d, J = 11.2 Hz,
1H), 5.13 (d, J = 9.2 Hz, 1H), 5.06−5.11 (m, 2H), 4.92−5.01 (m,
2H), 4.81−4.88 (m, 2H), 4.67 (d, J = 14.0 Hz, 1H), 4.35−4.4 (m,
2H), 3.79 (s, 3H), 3.47 (s, 3H), 3.36 (s, 3H), 3.22 (s, 3H), 3.18 (s,
3H), 3.06 (s, 3H), 2.69 (s, 3H), 2.66 (s, 3H), 2.31−2.39 (m, 1H),
2.23−2.29 (m, 1H), 2.11−2.18 (m, 2H), 1.93−2.04 (m, 4H), 1.55−
1.66 (m, 5H), 1.41−1.49 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J
= 6.4 Hz, 3H), 1.22 (d, J = 6.8 Hz, 3H), 0.99−1.01 (m, 8H), 0.94−
0.95 (m, 8H), 0.89−0.91 (m, 5H), 0.84−0.87 (m, 12H), 0.80 (d, J =
6.4 Hz, 6H), 0.62 (d, J = 6.8 Hz, 3H). HRMS (ESI): m/z [M + H]+
calcd for C67H113ClN11O13S 1346.7929, found 1346.7944.
1
that described for compound 9 in 19.1% yield in one step. H NMR
(400 MHz, CDCl3) δ 8.36 (d, J = 9.6 Hz, 1H), 8.01 (d, J = 6.4 Hz,
1H), 7.61 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.04 (d, J =
7.6 Hz, 1H), 6.99 (s, 1H), 6.81 (d, J = 8.0 Hz, 1H), 5.62−5.65 (m,
1H), 5.30−5.32 (m, 2H), 5.12−5.20 (m, 2H), 5.04−5.10 (m, 2H),
4.90−5.00 (m, 3H), 4.81−4.84 (m, 1H), 4.64 (d, J = 13.2 Hz, 1H),
4.33−4.38 (m, 1H), 4.28−4.30 (m, 1H), 3.86 (s, 3H), 3.84 (s, 3H),
3.47 (s, 3H), 3.37 (s, 3H), 3.20 (s, 3H), 3.15 (s, 3H), 3.04 (s, 3H),
2.67 (s, 3H), 2.64 (s, 3H), 2.27−2.35 (m, 2H), 2.06−2.12 (m, 3H),
1.89−2.03 (m, 3H), 1.54−1.66 (m, 5H), 1.37−1.49 (m, 3H), 1.31 (d,
J = 6.4 Hz, 3H), 1.23 (d, J = 6.0 Hz, 3H), 1.16 (d, J = 6.0 Hz, 3H),
0.95−0.99 (m, 9H), 0.89−0.93 (m, 10H), 0.82−0.87 (m, 13H),
0.79−0.80 (m, 4H), 0.73 (d, J = 6.0 Hz, 3H), 0.57 (d, J = 6.0 Hz,
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((4-
Chloro-3-methoxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-
e t h y l - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (40A). The title compound was obtained as a light yellow foam
using a method similar to that described for compound 9 in 19.1%
yield in one step. 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 10.0 Hz,
1H), 8.06 (d, J = 6.8 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.53 (d, J =
8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 2.0, 8.4 Hz, 1H),
7.02 (d, J = 2.0 Hz, 1H), 5.66 (dd, J = 4.0, 11.2 Hz, 1H), 5.44 (dd, J =
3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.6 Hz, 1H), 5.19 (d, J = 8.0 Hz,
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Article
1H), 5.17 (d, J = 9.6 Hz, 1H), 5.07−5.13 (m, 3H), 4.96−5.02 (m,
1H), 4.91−4.95 (m, 1H), 4.83−4.89 (m, 1H), 4.66 (d, J = 14.0 Hz,
1H), 4.33−4.42 (m, 2H), 3.90 (s, 3H), 3.48 (s, 3H), 3.39 (s, 3H),
3.22 (s, 3H), 3.18 (s, 3H), 3.07 (s, 3H), 2.70 (s, 3H), 2.66 (s, 3H),
2.38−2.46 (m, 1H), 2.26−2.35 (m, 1H), 2.11−2.17 (m, 3H), 1.96−
2.04 (m, 3H), 1.59−1.69 (m, 5H), 1.44−1.48 (m, 3H), 1.33 (d, J =
6.8 Hz, 3H), 1.25 (d, J = 6.8 Hz, 3H), 1.20 (d, J = 6.8 Hz, 3H), 1.00−
1.04 (m, 9H), 0.94−0.95 (m, 8H), 0.89−0.91 (m, 5H), 0.85−0.88
(m, 10H), 0.82 (d, J = 6.8 Hz, 4H), 0.75 (d, J = 6.8 Hz, 3H), 0.59 (d,
J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.76, 173.22,
173.17(2C), 171.45, 171.13, 170.88, 170.73, 170.34, 170.12, 168.59,
154.70, 136.13, 130.35, 123.06, 121.02, 114.14, 87.25, 82.17, 59.98,
58.03, 57.11, 56.25, 54.89, 54.55, 54.19, 50.04, 48.59, 48.22, 47.97,
44.55, 41.08, 40.78, 39.22, 39.12, 37.26, 36.33, 35.05, 32.54, 31.61,
31.28, 31.21, 29.93(3C), 29.62, 25.91, 24.83, 24.70, 24.48, 24.27,
23.77, 23.75, 23.66, 23.47, 23.37, 21.88, 21.39, 21.12, 20.54, 20.37,
18.82, 18.65, 18.56, 17.67, 14.88, 9.87. HRMS (ESI): m/z [M + H]+
calcd for C67H113ClN11O13S 1346.7929, found 1346.7924.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-(3-
(Benzyloxy)phenoxy)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (43A)
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5R)-5-(3-
(Benzyloxy)phenoxy)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (43B). Compound
9 (150 mg, 0.122 mmol) and 3-(benzyloxy)phenol (24.4 mg, 0.122
mmol) were dissolved in 4 mL of CH2Cl2. FeCl3 (10 mg, 0.061
mmol) and 4 Å molecular sieves (10 mg) were added to the solution,
stirred at room temperature for 16 h, and filtered. The filtrate was
evaporated to dryness, and the residue was purified by prep. HPLC to
give compound 43A (2 mg) and 43B (2 mg) as a white solid. Total
yield: 2.4%.
1
Compound 43A. H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6
Hz, 1H), 8.06 (d, J = 7.2 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.51 (d, J
= 8.4 Hz, 1H), 7.43 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 8.0 Hz, 3H), 7.32
(d, J = 7.2 Hz, 1H), 7.14 (t, J = 8.0 Hz, 1H), 6.59 (s, 2H), 5.69 (dd, J
= 4.0, 10.8 Hz, 1H), 5.62 (d, J = 5.2 Hz, 1H), 5.33 (dd, J = 3.6, 11.6
Hz, 1H), 5.22 (d, J = 9.2 Hz, 1H), 5.14 (d, J = 9.2 Hz, 1H), 5.09−
5.10 (m, 1H), 5.03 (s, 2H), 4.95−5.00 (m, 2H), 4.83−4.89 (m, 3H),
4.66 (d, J = 14.0 Hz, 1H), 4.39−4.44 (m, 1H), 4.33 (dd, J = 4.0, 9.6
Hz, 1H), 3.44 (s, 3H), 3.35 (s, 3H), 3.24 (s, 3H), 3.18 (s, 3H), 3.06
(s, 3H), 2.69 (s, 3H), 2.68 (s, 3H), 2.32−2.38 (m, 1H), 2.22−2.29
(m, 1H), 2.06−2.16 (m, 6H), 1.56−1.67 (m, 5H), 1.44−1.52 (m,
3H), 1.34 (d, J = 7.2 Hz, 3H), 1.28 (d, J = 7.2 Hz, 3H), 1.25 (d, J =
1.2 Hz, 3H), 0.98−1.03 (m, 12H), 0.93−0.95 (m, 9H), 0.86−0.91
(m, 14H), 0.82 (d, J = 6.4 Hz, 4H), 0.76 (d, J = 6.8 Hz, 3H). HRMS
(ESI): m/z [M + H]+ calcd for C73H118N11O14 1372.8860, found
1372.8803.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
(Benzyloxy)-4-chlorophenyl)thio)-3-methyltetrahydrofuran-2-yl)-
30-ethyl-6, 9, 18, 24-tetraisobutyl-3, 21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (41A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 24.2% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6 Hz, 1H),
8.08 (d, J = 6.8 Hz, 1H), 7.66 (d, J = 9.2 Hz, 1H), 7.53 (d, J = 8.0 Hz,
1H), 7.49 (d, J = 7.2 Hz, 2H), 7.36−7.40 (m, 3H), 7.29−7.33 (m,
1H), 7.13 (dd, J = 1.6, 8.0 Hz, 1H), 7.02 (d, J = 1.6 Hz, 1H), 5.66
(dd, J = 4.0, 11.2 Hz, 1H), 5.42 (dd, J = 4.0, 8.0 Hz, 1H), 5.33 (dd, J
= 4.0, 11.2 Hz, 1H), 5.23 (d, J = 9.2 Hz, 1H), 5.15−5.18 (m, 2H),
5.14 (s, 2H), 5.06−5.11 (m, 2H), 4.96−5.02 (m, 1H), 4.83−4.93 (m,
2H), 4.65 (d, J = 14.0 Hz, 1H), 4.36−4.43 (m, 1H), 4.31 (dd, J = 6.4,
9.2 Hz, 1H), 3.48 (s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H),
3.06 (s, 3H), 2.68 (s, 3H), 2.66 (s, 3H), 2.41−2.48 (m, 1H), 2.27−
2.35 (m, 1H), 2.07−2.17 (m, 3H), 1.97−2.03 (m, 3H), 1.56−1.69
(m, 5H), 1.41−1.49 (m, 3H), 1.31 (d, J = 7.2 Hz, 3H), 1.25 (d, J =
6.8 Hz, 3H), 1.15 (d, J = 6.8 Hz, 3H), 0.98−1.02 (m, 9H), 0.93−0.95
(m, 8H), 0.89−0.90 (m, 4H), 0.82−0.86 (m, 14H), 0.77 (d, J = 6.4
Hz, 4H), 0.62 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
173.66, 173.09, 173.05, 172.99, 171.29, 171.08, 170.82, 170.69,
170.28, 170.00, 168.52, 153.70, 136.31, 135.82, 130.50, 128.41(2C),
127.83, 127.06(2C), 123.35, 121.68, 115.85, 86.69, 81.86, 70.60,
59.33, 57.97, 57.01, 54.85, 54.55, 54.09, 49.95, 48.63, 48.24, 47.92,
44.56, 41.10, 40.58, 39.12, 38.95, 37.14, 36.19, 35.13, 32.58, 31.47,
31.20, 31.13, 29.83, 29.62, 29.58, 25.78, 24.74, 24.62, 24.49, 24.25,
23.67(3C), 23.47, 23.41, 23.30, 21.79, 21.29, 21.18, 20.25, 19.81,
18.83, 18.55, 18.50, 17.64, 15.02, 9.78. HRMS (ESI): m/z [M + H]+
calcd for C73H117ClN11O13S 1422.8242, found 1422.8207.
Compound 43B. 1H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 10.0
Hz, 1H), 7.93 (d, J = 6.8 Hz, 1H), 7.51 (d, J = 9.2 Hz, 1H), 7.44 (d, J
= 7.6 Hz, 1H), 7.34−7.39 (m, 3H), 7.27−7.33 (m, 2H), 7.09−7.14
(m, 1H), 6.62−6.63 (m, 1H), 6.55−6.59 (m, 2H), 5.66−5.69 (m,
1H), 5.60−5.62 (m, 1H), 5.31−5.35 (m, 1H), 5.28−5.29 (m, 1H),
5.18−5.22 (m, 1H), 5.03−5.07 (m, 2H), 5.02 (s, 2H), 4.93−4.98 (m,
1H), 4.82−4.87 (m, 2H), 4.69 (d, J = 7.6 Hz, 1H), 4.42−4.46 (m,
1H), 4.12−4.16 (m, 1H), 4.07−4.09 (m, 1H), 3.49 (s, 3H), 3.38 (s,
3H), 3.20 (s, 3H), 3.16 (s, 3H), 3.08 (s, 3H), 2.69 (s, 3H), 2.68 (s,
3H), 2.27−2.37 (m, 2H), 2.09−2.17 (m, 3H), 1.95−2.04 (m, 3H),
1.57−1.65 (m, 5H), 1.39−1.47 (m, 3H), 1.33 (d, J = 7.2 Hz, 5H),
1.23−1.24 (m, 2H), 1.16−1.20 (m, 2H), 1.02−1.04 (m, 4H), 0.98−
0.99 (m, 7H), 0.92−0.95 (m, 9H), 0.87−0.90 (m, 8H), 0.84 (d, J =
7.2 Hz, 4H), 0.79 (d, J = 6.8 Hz, 4H), 0.76 (d, J = 6.0 Hz, 3H), 0.59
(d, J = 6.0 Hz, 3H). HRMS (ESI): m/z [M + H]+ calcd for
C73H118N11O14 1372.8860, found 1372.8825.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-(3,5-Di-
methoxyphenoxy)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (44A). The title
compound was obtained as a white solid using a method similar to
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((2,6-
Dimethoxyphenyl)thio)-3-methyltetrahydrofuran-2-yl)-30-ethyl-
6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-
nonamethyl-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritria-
contan-2,5,8,11,14,17,20,23,26,29,32-undecaone (42A). The title
compound was obtained as a white solid using a method similar to
1
that described for compound 43A in 10.1% yield in one step. H
1
that described for compound 9 in 3.7% yield in one step. H NMR
NMR (400 MHz, CDCl3) δ 8.34 (d, J = 9.2 Hz, 1H), 8.04 (d, J = 6.0
Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 6.08−
6.17 (m, 1H), 6.01 (s, 1H), 5.95 (s, 1H), 5.64−5.69 (m, 1H), 5.33−
5.39 (m, 2H), 5.20−5.29 (m, 2H), 4.98−5.14 (m, 5H), 4.83−4.88
(m, 1H), 4.67−4.74 (m, 1H), 4.36−4.42 (m, 2H), 3.74 (s, 3H), 3.71
(s, 3H), 3.51 (s, 3H), 3.37 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s,
3H), 2.69 (s, 3H), 2.67 (s, 3H), 2.28−2.41 (m, 3H), 2.06−2.20 (m,
5H), 1.91−2.00 (m, 3H), 1.56−1.72 (m, 4H), 1.41−1.49 (m, 2H),
1.34 (d, J = 6.8 Hz, 3H), 1.24−1.27 (m, 3H), 1.12 (d, J = 6.0 Hz,
2H), 0.97−1.03 (m, 5H), 0.90−0.95 (m, 13H), 0.80−0.86 (m, 15H),
0.65−0.69 (m, 2H), 0.63 (d, J = 6.4 Hz, 4H), 0.52 (d, J = 6.4 Hz,
3H). HRMS (ESI): m/z [M + H]+ calcd for C68H116N11O15
1326.8652, found 1326.8628.
(400 MHz, CDCl3) δ 8.10 (d, J = 9.6 Hz, 1H), 7.97 (d, J = 6.4 Hz,
1H), 7.70 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.28−7.33
(m, 2H), 7.18−7.22 (m, 1H), 5.67−5.71 (m, 1H), 5.43−5.50 (m,
1H), 5.31−5.34 (m, 1H), 5.28−5.30 (m, 1H), 5.21−5.25 (m, 1H),
5.08−5.13 (m, 2H), 5.04−5.07 (m, 1H), 4.85−4.88 (m, 1H), 4.81−
4.84 (m, 1H), 4.74 (t, J = 8.8 Hz, 1H), 4.66 (d, J = 14.0 Hz, 1H),
4.48−4.52 (m, 1H), 4.42−4.46 (m, 1H), 3.82 (s, 6H), 3.50 (s, 3H),
3.24 (s, 3H), 3.15 (s, 3H), 3.07 (s, 3H), 3.02 (s, 3H), 2.71 (s, 6H),
2.42−2.48 (m, 1H), 2.26−2.34 (m, 2H), 2.08−2.15 (m, 5H), 1.97−
2.01 (m, 3H), 1.84−1.89 (m, 5H), 1.55−1.60 (m, 3H), 1.44−1.48
(m, 3H), 1.30−1.33 (m, 3H), 1.24−1.27 (m, 5H), 1.14−1.17 (m,
2H), 1.03−1.06(m, 4H), 0.94−1.00 (m, 14H), 0.84−0.88 (m, 13H),
0.73−0.76 (m, 4H). HRMS (ESI): m/z [M + H]+ calcd for
C68H116N11O14S 1342.8424, found 1342.8396.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-((2-fluorobenzyl)oxy)phenyl)thio)-3-methyltetrahydrofuran-
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Article
2 - y l ) - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (45A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 22.8% yield in
2.28−2.33 (m, 1H), 2.08−2.16 (m, 3H), 1.93−2.04 (m, 3H), 1.58−
1.70 (m, 5H), 1.39−1.50 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J
= 6.8 Hz, 3H), 1.17 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.4 Hz, 3H),
0.96−0.99 (m, 6H), 0.93−0.95 (m, 7H), 0.89−0.91 (m, 4H), 0.85−
0.88 (m, 12H), 0.81 (d, J = 6.8 Hz, 4H), 0.78 (d, J = 6.4 Hz, 3H),
0.62 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.65,
173.10, 173.07, 173.00, 171.39, 171.11, 170.81, 170.70, 170.30,
169.81, 168.62, 163.63, 163.17, 158.38, 137.38, 132.59, 129.87,
129.35, 129.27, 123.00, 116.46, 115.46, 115.24, 113.60, 86.65, 81.96,
69.19, 59.70, 57.85, 56.99, 54.88, 54.54, 54.15, 49.99, 48.64, 48.29,
47.94, 44.60, 41.16, 40.72, 39.18, 39.00, 37.16, 36.23, 35.11, 32.62,
31.55, 31.24, 31.15, 29.86, 29.70, 29.61, 25.84, 24.78, 24.65, 24.56,
24.34, 23.79, 23.69, 23.50, 23.44, 23.33, 21.85, 21.34, 21.13, 20.18,
20.10, 18.84, 18.60, 18.54, 17.67, 15.01, 9.81. HRMS (ESI): m/z [M
+ H]+ calcd for C73H117FN11O13S 1406.8537, found 1406.8511.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-((2-
Chloro-4-fluorobenzyl)oxy)phenyl)thio)-3-methyltetrahydrofuran-
2-yl)-30-ethyl-6,9,18,24-tetraisobutyl-3,21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (48A). The title compound was obtained as a light yellow solid
using a method similar to that described for compound 9 in 14.3%
yield in one step. 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 9.6 Hz,
1H), 8.05 (d, J = 6.8 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.55 (d, J =
6.4 Hz, 1H), 7.49−7.54 (m, 2H), 7.13−7.18 (m, 2H), 7.01−7.03 (m,
2H), 6.81 (dd, J = 2.0, 8.0 Hz, 1H), 5.66 (dd, J = 4.0, 11.2 Hz, 1H),
5.50 (dd, J = 3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.2 Hz, 1H), 5.17−
5.22 (m, 2H), 5.12−5.14 (m, 1H), 5.10 (s, 2H), 5.05−5.08 (m, 2H),
4.96−5.02 (m, 1H), 4.92 (t, J = 8.4 Hz, 1H), 4.84 (t, J = 7.6 Hz, 1H),
4.65 (d, J = 13.6 Hz, 1H), 4.37−4.42 (m, 1H), 4.32 (dd, J = 6.4, 9.2
Hz, 1H), 3.46 (s, 3H), 3.37 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06
(s, 3H), 2.67 (s, 3H), 2.65 (s, 3H), 2.42−2.48 (m, 1H), 2.28−2.33
(m, 1H), 2.08−2.14 (m, 3H), 1.93−2.03 (m, 3H), 1.54−1.69 (m,
6H), 1.43−1.49 (m, 2H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J = 7.2
Hz, 3H), 1.17 (d, J = 6.4 Hz, 3H), 0.97−1.02 (m, 8H), 0.93−0.96 (m,
8H), 0.89−0.90 (m, 4H), 0.84−0.87 (m, 12H), 0.81 (d, J = 6.4 Hz,
4H), 0.78 (d, J = 6.8 Hz, 3H), 0.63 (d, J = 6.4 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 173.67, 173.10, 173.00, 171.46, 171.14, 170.82,
170.73, 170.31, 169.81, 168.63, 158.14, 137.56, 130.17, 130.08,
129.98, 123.18, 116.88, 116.63, 116.60, 114.21, 114.01, 113.36, 86.62,
81.88, 66.58, 59.80, 57.87, 57.04, 54.91, 54.55, 54.17, 50.02, 48.63,
48.29, 47.96, 44.60, 41.23, 40.75, 39.21, 39.07, 37.21, 36.27, 35.17,
32.64, 31.60, 31.28, 31.19, 29.89, 29.75, 29.63, 25.89, 24.80,
24.67(2C), 24.59, 24.36, 23.83, 23.72(2C), 23.59, 23.47, 23.36,
21.89, 21.37, 21.17, 20.22(2C), 18.86, 18.63, 18.57, 17.69, 14.97,
9.83. HRMS (ESI): m/z [M + H]+calcd for C73H116ClFN11O13S
1440.8147, found 1440.8098.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-((3-
Chloro-4-fluorobenzyl)oxy)phenyl)thio)-3-methyltetrahydrofuran-
2-yl)-30-ethyl-6,9,18,24-tetraisobutyl-3,21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (49A). The title compound was obtained as a light yellow solid
using a method similar to that described for compound 9 in 38.6%
yield in one step. 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.6 Hz,
1H), 8.05 (d, J = 6.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.55 (dd, J =
2.0, 6.8 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.30−7.33 (m, 1H), 7.24
(d, J = 8.0 Hz, 1H), 7.14−7.16 (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 1.6,
2.4 Hz, 1H), 6.80 (ddd, J = 0.8, 2.8, 7.6 Hz, 1H), 5.65 (dd, J = 4.0,
10.8 Hz, 1H), 5.48 (dd, J = 3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.2
Hz, 1H), 5.21 (d, J = 9.6 Hz, 1H), 5.19 (d, J = 11.2 Hz, 1H), 5.02−
5.14 (m, 3H), 5.00 (d, J = 2.8 Hz, 2H), 4.96−4.98 (m, 1H), 4.91 (t, J
= 8.0 Hz, 1H), 4.85 (t, J = 8.0 Hz, 1H), 4.66 (d, J = 14.0 Hz, 1H),
4.37−4.42 (m, 1H), 4.33 (dd, J = 6.0, 9.2 Hz, 1H), 3.47 (s, 3H), 3.38
(s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s, 3H), 2.69 (s, 3H), 2.66 (s,
3H), 2.39−2.47 (m, 1H), 2.27−2.35 (m, 1H), 2.11−2.17 (m, 3H),
1.96−2.04 (m, 3H), 1.59−1.69 (m, 5H), 1.42−1.49 (m, 3H), 1.33 (d,
J = 7.2 Hz, 3H), 1.25 (d, J = 6.8 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H),
1.01 (d, J = 6.4 Hz, 3H), 0.97 (d, J = 2.8 Hz, 3H), 0.96 (d, J = 3.2 Hz,
3H), 0.93−0.95 (m, 7H), 0.89−0.91 (m, 4H), 0.85−0.88 (m, 12H),
0.81 (d, J = 6.4 Hz, 4H), 0.77 (d, J = 6.4 Hz, 3H), 0.60 (d, J = 6.4 Hz,
1
one step. H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 9.6 Hz, 1H),
8.06 (d, J = 6.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.49−7.53 (m,
2H), 7.29−7.32 (m, 1H), 7.14−7.18 (m, 3H), 7.05−7.10 (m, 1H),
7.02 (t, J = 2.0 Hz, 1H), 6.83 (dd, J = 2.4, 8.0 Hz, 1H), 5.66 (dd, J =
4.0, 11.2 Hz, 1H), 5.50 (dd, J = 3.6, 7.6 Hz, 1H), 5.33 (dd, J = 4.0,
11.2 Hz, 1H), 5.18−5.23 (m, 2H), 5.12 (s, 2H), 5.02−5.11 (m, 3H),
4.96−5.00 (m, 1H), 4.90−4.95 (m, 1H), 4.83−4.87 (m, 1H), 4.66 (d,
J = 13.6 Hz, 1H), 4.37−4.41 (m, 1H), 4.32 (dd, J = 6.4, 9.6 Hz, 1H),
3.47 (s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s, 3H),
2.68 (s, 3H), 2.66 (s, 3H), 2.40−2.48 (m, 1H), 2.28−2.34 (m, 1H),
2.09−2.17 (m, 3H), 1.96−2.05 (m, 3H), 1.57−1.69 (m, 5H), 1.42−
1.51 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J = 6.8 Hz, 3H), 1.18
(d, J = 6.8 Hz, 3H), 0.97−1.02 (m, 9H), 0.93−0.95 (m, 8H), 0.89−
0.91 (m, 4H), 0.85−0.88 (m, 11H), 0.81 (d, J = 6.4 Hz, 4H), 0.79 (d,
J = 6.4 Hz, 3H), 0.63 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/z [M +
H]+ calcd for C73H117FN11O13S 1406.8537, found 1406.8514.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-((3-fluorobenzyl)oxy)phenyl)thio)-3-methyltetrahydrofuran-
2 - y l ) - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (46A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 53.0% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 10.0 Hz, 1H),
8.05 (d, J = 6.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 7.6 Hz,
1H), 7.31−7.35 (m, 1H), 7.19−7.21 (m, 2H), 7.14−7.17 (m, 2H),
6.98−7.02 (m, 2H), 6.80−6.82 (m, 1H), 5.66 (dd, J = 4.0, 10.8 Hz,
1H), 5.49 (dd, J = 3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.2 Hz, 1H),
5.18−5.23 (m, 2H), 5.07−5.14 (m, 3H), 5.05 (s, 2H), 4.98−5.02 (m,
1H), 4.92 (t, J = 8.4 Hz, 1H), 4.85 (t, J = 6.4 Hz, 1H), 4.66 (d, J =
13.6 Hz, 1H), 4.37−4.42 (m, 1H), 4.32 (dd, J = 6.4, 9.2 Hz, 1H), 3.47
(s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.18 (s, 3H), 3.06 (s, 3H), 2.68 (s,
3H), 2.66 (s, 3H), 2.40−2.48 (m, 1H), 2.28−2.34 (m, 1H), 2.08−
2.17 (m, 3H), 1.96−2.03 (m, 3H), 1.58−1.70 (m, 5H), 1.40−1.50
(m, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.25 (d, J = 7.2 Hz, 3H), 1.18 (d, J
= 6.8 Hz, 3H), 1.01 (d, J = 6.4 Hz, 3H), 0.96−0.99 (m, 7H), 0.93−
0.95 (m, 8H), 0.89−0.91 (m, 4H), 0.85−0.88 (m, 10H), 0.82 (d, J =
6.4 Hz, 4H), 0.78 (d, J = 6.8 Hz, 3H), 0.62 (d, J = 6.4 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 173.65, 173.10, 173.04, 173.03, 171.37,
171.11, 170.81, 170.69, 170.30, 169.82, 168.62, 158.28, 137.42,
130.00, 129.85, 123.12, 122.70, 122.68, 116.45, 114.76, 114.55,
114.33, 114.11, 113.62, 86.66, 81.99, 69.03, 69.01, 59.62, 57.86, 56.99,
54.88, 54.54, 54.14, 49.98, 48.66, 48.28, 47.94, 44.60, 41.15, 40.67,
38.97, 37.15, 36.21, 35.10, 32.63, 31.53, 31.22, 31.13, 29.85, 29.83,
29.60, 25.82, 24.77, 24.64, 24.55, 24.32, 23.76, 23.67, 23.47, 23.42,
23.30, 21.83, 21.33, 21.14, 20.17, 20.05, 18.83, 18.59, 18.53, 17.67,
15.03, 9.80. HRMS (ESI): m/z [M + H]+ calcd for C73H117FN11O13S
1406.8537, found 1406.8513.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-((4-fluorobenzyl)oxy)phenyl)thio)-3-methyltetrahydrofuran-
2 - y l ) - 6 , 9 , 1 8 , 2 4 - t e t r a i s o b u t y l - 3 , 2 1 - d i i s o p r o p y l -
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (47A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 47.0% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 10.0 Hz, 1H),
8.05 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.4 Hz,
1H), 7.41 (dd, J = 5.6, 8.8 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.15 (d,
J = 8.0 Hz, 1H), 7.06 (t, J = 8.8 Hz, 2H), 7.00 (t, J = 2.0 Hz, 1H),
6.80 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 5.66 (dd, J = 4.0, 11.2 Hz, 1H),
5.49 (dd, J = 3.6, 8.0 Hz, 1H), 5.33 (dd, J = 4.0, 11.2 Hz, 1H), 5.21
(d, J = 7.2 Hz, 1H), 5.19 (d, J = 8.4 Hz, 1H), 5.06−0.14 (m, 3H),
5.01 (s, 2H), 4.96−4.98 (m, 1H), 4.92 (t, J = 8.0 Hz, 1H), 4.85 (t, J =
8.0 Hz, 1H), 4.66 (d, J = 14.0 Hz, 1H), 4.37−4.42 (m, 1H), 4.32 (dd,
J = 6.4, 9.6 Hz, 1H), 3.46 (s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.17 (s,
3H), 3.06 (s, 3H), 2.69 (s, 3H), 2.66 (s, 3H), 2.39−2.47 (m, 1H),
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Article
3H). 13C NMR (100 MHz, CDCl3) δ 173.85, 173.31, 173.22(2C),
171.53, 171.31, 171.01, 170.88, 170.51, 170.03, 168.81, 159.01,
158.35, 156.54, 137.65, 134.18, 130.05, 129.92, 127.39, 127.32,
123.52, 116.80, 116.59, 113.92, 86.89, 82.23, 68.66, 59.77, 58.07,
57.17, 55.07, 54.76, 54.33, 50.18, 48.86, 48.47, 48.14, 44.80, 41.30,
40.86, 39.35, 39.16, 37.32, 36.40, 35.27, 32.81, 31.71, 31.41, 31.33,
30.04, 29.86, 29.80, 26.00, 24.95, 24.82, 24.73, 24.50, 23.95, 23.86,
23.65, 23.62, 23.49, 22.01, 21.52, 21.31, 20.37, 20.26, 19.03, 18.78,
18.71, 17.85, 15.23, 9.99. HRMS (ESI): m/z [M + H]+ calcd for
C73H116ClFN11O13S 1440.8147, found 1440.8121.
mixture 52m (50 mg, major: minor = 1:1.4) were obtained as a white
solid using a method similar to that described for compound 9 in total
42.9% yield in one step. H NMR (400 MHz, CDCl3) δ 8.37 (d, J =
1
10.0 Hz, 1H), 8.05 (d, J = 6.8 Hz, 1H), 7.57 (d, J = 9.2 Hz, 1H), 7.52
(d, J = 8.4 Hz, 1H), 7.41 (dd, J = 5.2, 8.4 Hz, 2H), 7.05 (t, J = 8.8 Hz,
2H), 6.71 (dd, J = 1.2, 2.0 Hz, 1H), 6.66 (dd, J = 1.2, 2.0 Hz, 1H),
6.37 (t, J = 2.4 Hz, 1H), 5.63 (dd, J = 4.0, 11.2 Hz, 1H), 5.44 (dd, J =
3.2, 8.0 Hz, 1H), 5.32 (dd, J = 4.0, 11.6 Hz, 1H), 5.18 (d, J = 10.8 Hz,
1H), 5.12 (d, J = 9.2 Hz, 1H), 5.06−5.10 (m, 1H), 5.00−5.06 (m,
2H), 4.99 (s, 2H), 4.90−4.96 (m, 2H), 4.80−4.87 (m, 1H), 4.66 (d, J
= 14.0 Hz, 1H), 4.36−4.40 (m, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 3.36
(s, 3H), 3.21 (s, 3H), 3.17 (s, 3H), 3.05 (s, 3H), 2.67 (s, 3H), 2.64 (s,
3H), 2.27−2.38 (m, 2H), 2.08−2.17 (m, 3H), 1.92−1.99 (m, 3H),
1.58−1.68 (m, 5H), 1.39−1.49 (m, 3H), 1.32 (d, J = 7.2 Hz, 3H),
1.25 (d, J = 6.8 Hz, 3H), 1.20 (d, J = 7.2 Hz, 3H), 1.00 (d, J = 6.8 Hz,
3H), 0.94−0.97 (m, 8H), 0.92−0.94 (m, 4H), 0.88−0.91 (m, 5H),
0.85−0.87 (m, 8H), 0.83−0.84 (m, 4H), 0.80 (d, J = 6.8 Hz, 4H),
0.76 (d, J = 6.4 Hz, 3H), 0.57 (d, J = 6.4 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 173.69, 173.31, 173.13, 173.11, 171.32, 171.09,
170.86, 170.66, 170.38, 169.91, 168.72, 166.68, 163.62, 161.17,
160.69, 159.43, 137.65, 132.58, 132.55, 129.40, 129.32, 115.44,
115.23, 109.72, 108.79, 100.91, 87.48, 82.92, 69.28, 59.97, 57.87,
56.94, 55.48, 54.84, 54.53, 54.17, 49.98, 48.74, 48.24, 47.98, 44.60,
40.84, 40.46, 39.18, 38.95, 37.12, 36.21, 34.74, 32.71, 31.50, 31.18,
31.10, 29.83, 29.61, 25.80, 24.79, 24.64, 24.47, 24.24, 23.69, 23.62,
23.49, 23.42, 23.29, 21.82, 21.35, 21.11, 20.60, 20.23, 18.68, 18.51,
17.68, 15.06, 9.89. HRMS (ESI): m/z [M + H]+ calcd for
C74H119FN11O14S 1436.8643, found 1436.8613.
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-((4-fluoro-2-methylbenzyl)oxy)phenyl)thio)-3-methyltetrahy-
drofuran-2-yl)-6,9,18,24-tetraisobutyl-3,21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (50A). The title compound was obtained as a white solid using
a method similar to that described for compound 9 in 49.3% yield in
1
one step. H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 10.0 Hz, 1H),
8.04 (d, J = 6.0 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.50 (d, J = 7.2 Hz,
1H), 7.35−7.38 (m, 1H), 7.23−7.26 (m, 1H), 7.14−7.20 (m, 1H),
7.00−7.02 (m, 1H), 6.79−6.97 (m, 3H), 5.63−5.68 (m, 1H), 5.48−
5.53 (m, 1H), 5.30−5.34 (m, 1H), 5.18−5.23 (m, 2H), 5.05−5.14
(m, 2H), 4.99−5.01 (m, 1H), 4.96 (s, 2H), 4.82−4.93 (m, 3H), 4.65
(d, J = 13.6 Hz, 1H), 4.36−4.42 (m, 1H), 4.30−4.34 (m, 1H), 3.46
(s, 3H), 3.38 (s, 3H), 3.22 (s, 3H), 3.17 (s, 3H), 3.06 (s, 3H), 2.69 (s,
3H), 2.66 (s, 3H), 2.39−2.49 (m, 1H), 2.35 (s, 3H), 2.23−2.33 (m,
1H), 2.07−2.18 (m, 3H), 1.90−2.05 (m, 3H), 1.54−1.73 (m, 5H),
1.38−1.52 (m, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.25 (d, J = 6.8 Hz,
3H), 1.17 (d, J = 6.8Hz, 3H), 0.96−1.02 (m, 9H), 0.90−0.94 (m,
10H), 0.84−0.88 (m, 13H), 0.77−0.82 (m, 7H), 0.63 (d, J = 6.0 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 173.67, 173.13, 173.11, 173.00,
171.46, 171.14, 170.82, 170.74, 170.31, 169.83, 168.64, 158.52,
137.45, 130.61, 130.52, 130.42, 129.96, 122.90, 117.23, 117.02,
116.31, 113.49, 112.60, 112.39, 86.62, 81.90, 67.93, 59.80, 57.87,
57.04, 54.91, 54.56, 54.17, 50.01, 48.62, 48.29, 47.95, 44.60, 41.23,
40.78, 39.22, 39.07, 37.22, 36.28, 35.17, 32.63, 31.61, 31.28, 31.19,
30.94, 29.90, 29.77, 29.63, 25.89, 24.81, 24.68, 24.60, 24.36, 23.84,
23.72, 23.59, 23.47, 23.38, 21.89, 21.37, 21.15, 20.22, 19.00, 18.88,
18.62, 18.57, 17.69, 14.97, 9.83. HRMS (ESI): m/z [M + H]+ calcd
for C74H119FN11O13S 1420.8694, found 1420.8663.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01484.
Concentration in plasma of preliminary pharmacoki-
netics experiment (PO) data of the selected compounds;
plasma protein binding study of the compound 27A;
2D-NMR analysis of 27A and 27B; inhibition of HBV
infection by 27A and 27B on primary human
hepatocytes (PHH); fluorescence intensity analysis of
Figure 4B,C; treatment of C57B/L mice by 27A of
indicated dosage (n = 5 for each group) for 14 days;
inhibition of HBV virus infection of HepG2-NTCP cells
by 27A and Alisporivir; synthesis of mercapto hetero-
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-33-((2R,3R,5S)-5-((3-
Chloro-5-((4-fluorobenzyl)oxy)phenyl)thio)-3-methyltetrahydrofur-
an-2-yl)-30-ethyl-6,9,18,24-tetraisobutyl-3,21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (51A). The title compound 51A (major, 12 mg) and the
mixture 50m (15 mg, major:minor = 3:1) were obtained as a white
solid using a method similar to that described for compound 9 in total
1
cyclic and aromatic intermediates (1−28); and H and
13C NMR spectra of compounds (PDF)
Molecular formula strings (CSV)
1
11.6% yield in one step. H NMR (400 MHz, CDCl3) δ 8.37 (d, J =
9.6 Hz, 1H), 8.04 (d, J = 6.8 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.53
(d, J = 8.0 Hz, 1H), 7.42 (dd, J = 5.6 Hz, 1H), 7.40 (d, J = 5.6 Hz,
1H), 7.04−7.09 (m, 3H), 6.88−6.92 (m, 1H), 6.78−6.81 (m, 1H),
5.65 (dd, J = 4.0, 11.2 Hz, 1H), 5.48 (dd, J = 2.8, 7.6 Hz, 1H), 5.32
(dd, J = 3.6, 11.2 Hz, 1H), 5.20 (d, J = 10.8 Hz, 1H), 5.16 (d, J = 8.8
Hz, 1H), 5.04−5.11 (m, 2H), 5.00 (s, 2H), 4.94−4.98 (m, 2H),
4.90−4.92 (m, 1H), 4.81−4.88 (m, 1H), 4.66 (d, J = 13.6 Hz, 1H),
4.34−4.44 (m, 2H), 3.47 (s, 3H), 3.35 (s, 3H), 3.22 (s, 3H), 3.17 (s,
3H), 3.06 (s, 3H), 2.67 (s, 3H), 2.65 (s, 3H), 2.33−2.41 (m, 1H),
2.24−2.31(m, 1H), 2.10−2.18 (m, 3H), 1.94−2.04 (m, 3H), 1.55−
1.68 (m, 5H), 1.39−1.46 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.25 (d, J
= 6.8 Hz, 3H), 1.19 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.4 Hz, 3H),
0.93−0.97 (m, 12H), 0.89−0.91 (m, 6H), 0.85−0.86 (m, 11H), 0.83
(d, J = 3.2 Hz, 3H), 0.79−0.81 (m, 4H), 0.64 (d, J = 6.8 Hz, 3H).
HRMS (ESI): m/z [M + H]+ calcd for C73H116ClFN11O13S
1440.8147, found 1440.8153.
AUTHOR INFORMATION
■
Corresponding Authors
Zhiyuan Zhang − National Institute of Biological Sciences,
Beijing 102206, China; orcid.org/0000-0002-2910-
1986; Phone: +86-10-80726688-8575;
Email: zhangzhiyuan@nibs.ac.cn
Wenhui Li − National Institute of Biological Sciences, Beijing
102206, China; Email: liwenhui@nibs.ac.cn
Authors
Yang Liu − National Institute of Biological Sciences, Beijing
102206, China; Graduate Program, Tsinghua University,
Beijing 100084, China
Hanying Ruan − National Institute of Biological Sciences,
Beijing 102206, China
Ying Li − National Institute of Biological Sciences, Beijing
102206, China
(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-((2R,3R,5S)-
5-((3-((4-fluorobenzyl)oxy)-5-methoxyphenyl)thio)-3-methyltetra-
hydrofuran-2-yl)-6,9,18,24-tetraisobutyl-3,21-diisopropyl-
1,4,7,10,12,15,19,25,28-nonamethyl-1,4,7,10,13,16,19,22,25,28,31-
undecaazacyclotritriacontan-2,5,8,11,14,17,20,23,26,29,32-unde-
caone (52A). The title compound 52A (major, 50 mg) and the
563
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Article
Guoliang Sun − National Institute of Biological Sciences,
Beijing 102206, China; Graduate Program, Peking
University, Beijing 100080, China
porting polypeptide; CFSE, carboxyfluorescein succinimidyl
ester; PMA, phorbol myristate acetate
Xiao Liu − National Institute of Biological Sciences, Beijing
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Wenhui He − National Institute of Biological Sciences, Beijing
102206, China
Fengfeng Mao − National Institute of Biological Sciences,
Beijing 102206, China
Miaomiao He − National Institute of Biological Sciences,
Beijing 102206, China; Graduate Program, Chinese
Academy of Medical Sciences and Peking Union Medical
College, Beijing 100005, China
Liwei Yan − National Institute of Biological Sciences, Beijing
102206, China
Guocai Zhong − National Institute of Biological Sciences,
Beijing 102206, China
Huan Yan − National Institute of Biological Sciences, Beijing
102206, China
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01484
Author Contributions
Y.L. and H.R. contributed equally. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript. Z.Z. and W.L.
conceived the project; Z.Z. and H.R. designed the project;
H.R. and Y.L. synthesized all of the compounds; W.L. and Y.L.
designed the project; Y.L. conducted the biological experi-
ments in vitro and in vivo; X.L. tested liver microsomes
stability, prepared compounds formulation, and tested
pharmacokinetic parameters; G.S. helped with biological
experiments in vitro; W.H. and F.M. helped with mice
experiments; M.H. conducted mice spleen T cells assays;
L.Y. produced HBV and HDV inoculum for antiviral assays;
G.Z. and H.Y. conducted IC50 determine assays of several
compounds; and Y.L. and H.R. drafted the manuscript.
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Funding
(11) Yan, H.; Peng, B.; He, W.; Zhong, G.; Qi, Y.; Ren, B.; Gao, Z.;
Jing, Z.; Song, M.; Xu, G.; Sui, J.; Li, W. Molecular determinants of
hepatitis B and D virus entry restriction in mouse sodium
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7991.
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J.; Li, W. Viral entry of hepatitis B and D viruses and bile salts
transportation share common molecular determinants on sodium
taurocholate cotransporting polypeptide. J. Virol. 2014, 88, 3273−
3284.
This project was funded by the National Natural Science
Foundation of China to W.L. (81525018); the Science and
Technology Major Project of Beijing to W.L.
(D171100003117003); the Beijing Scholar Project to W.L.;
and the Science and Technology Bureau of Beijing Municipal
Government to W.L.
Notes
The authors declare no competing financial interest.
̈
(13) Konig, A.; Doring, B.; Mohr, C.; Geipel, A.; Geyer, J.; Glebe, D.
ACKNOWLEDGMENTS
■
Kinetics of the bile acid transporter and hepatitis B virus receptor Na
+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes. J.
Hepatol. 2014, 61, 867−875.
The authors thank Metabolomic Center of National Institute
of Biological Sciences for assistance with LC−MS analysis,
Chemistry Center of National Institute of Biological Sciences
for assistance with 2D-NMR analysis, and Tsinghua University
for Ph.D. program support.
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I.; Chen, C.-J. The rs2296651 (S267F) variant on NTCP (SLC10A1)
is inversely associated with chronic hepatitis B and progression to
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ABBREVIATIONS
■
HBV, Hepatitis B virus; HDV, Hepatitis D virus; NTCP,
Sodium taurocholate co-transporting peptide; CsA, Cyclo-
sporine A; TEA, triethylamine; TMSOTf, trimethylsilyl
trifluoromethanesulfonate; BSA, N,O-bis(trimethylsilyl)-
acetamide; ACN, acetonitrile; SAR, structure−activity relation-
ship; DMSO, dimethyl sulfoxide; TCA, taurocholic acid;
TLCA, taurolithocholic acid; OATP, organic anion-trans-
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