Synthesis and biological evaluation of 2-epi-jaspine B analogs as selective sphingosine kinase 1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103369

2-Epi-jaspine B is an isomer of the natural product jaspine B and shows certain selectivity for SphK1 and potent antitumor activity. Based on the crystal structure of SphK1, we transformed the structure of 2-epi-jaspine B and modified the hydrophobic side chain to obtain a series of 2-epi-jaspine B analogs. The MTT assay was used to examine the antitumor activities of these analogs. We identified a novel 2-epi-jaspine B analog YHR17, which has potent antiproliferative activities for tested cell lines with IC₅₀ values that ranged from 0.68 to 5.68 μM and inhibited the proliferation of the A375 cell line by affecting the cell cycle and apoptosis. Furthermore, YHR17 inhibited SphK1 with more than 125-fold selectivity over SphK2.

Full text of DOI:10.1016/j.bioorg.2019.103369

Journal Pre-proofs
Synthesis and biological evaluation of 2-epi-jaspine B analogs as selective
sphingosine kinase 1 inhibitors
Haoran Yang, Ying Li, Huining Chai, Takayuki Yakura, Bo Liu, Qingqiang
Yao
PII:
S0045-2068(19)31138-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.103369
YBIOO 103369
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
18 July 2019
4 October 2019
14 October 2019
Please cite this article as: H. Yang, Y. Li, H. Chai, T. Yakura, B. Liu, Q. Yao, Synthesis and biological evaluation
of 2-epi-jaspine B analogs as selective sphingosine kinase 1 inhibitors, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103369
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Graphical Abstract
A tetrahydropyrrolidine analog YHR17, which was obtained using structural
transformation and modification of 2-epi-jaspine B, inhibited SphK1 with more than
125-fold selectivity over SphK2. This analog demonstrated potent antiproliferative
activities with IC50 values ranging from 0.68 to 5.68 μM against tested cancer cell lines
and inhibited proliferation of the A375 cell line by affecting cell cycle and apoptosis.
tetrahydrofuran ring is modified to
tetrahydropyrrole ring;
C2 link large hydrophobic group;
H2N
OH
2
H2N
OH
2
4
4
O
11
N
anticancer activity enhanced;
SphK1 selectivity improved.
C14H29
O
H
O
2-epi-jaspine B
YHR17
IC50 SphK1   M
IC50 SphK2   M
Selectivity(SphK2SphK1)  
IC50 HepG2   M
IC50 HCFB  M
IC50 SphK1   M
IC50 SphK2  M
Selectivity(SphK2SphK1) 
IC50 HepG2   
IC50 HCFB  M

Title page
Title:
Synthesis and biological evaluation of 2-epi-jaspine B analogs as selective sphingosine
kinase 1 inhibitors
Author names and affiliations:
Haoran Yang1,2,3#, Ying Li1,2,3#, Huining Chai1,2,3, Takayuki Yakura4, Bo Liu1,2,3*,
Qingqiang Yao1,2,3
*
1Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062,
P. R. China; 2Key Laboratory for Biotech-Drugs Ministry of Health, Jinan 250062, P.
3
R. China; Key Laboratory for Rare & Uncommon Diseases of Shandong Province,
4
Jinan 250062, P. R. China; Graduate School of Medicine and Pharmaceutical
Sciences, University of Toyama, Toyama 930-0194, Japan.
* Corresponding address:
Tel.: +86-531-82919962; E-mail: mls_liub@ujn.edu.cn; yao_imm@163.com
#: These authors contributed equally.
Abstract:
2-Epi-jaspine B is an isomer of the natural product jaspine B and shows certain
selectivity for SphK1 and potent antitumor activity. Based on the crystal structure of
SphK1, we transformed the structure of 2-epi-jaspine B and modified the hydrophobic
side chain to obtain a series of 2-epi-jaspine B analogs. The MTT assay was used to
examine the antitumor activities of these analogs. We identified a novel 2-epi-jaspine
B analog YHR17, which has potent antiproliferative activities for tested cell lines with
IC50 values that ranged from 0.68 to 5.68 μM and inhibited the proliferation of the A375
cell line by affecting the cell cycle and apoptosis. Furthermore, YHR17 inhibited
SphK1 with more than 125-fold selectivity over SphK2.
Keywords: SphK1, SphK1 inhibitors, 2-epi-jaspine B, tetrahydropyrrolidine, anticancer.

1. Introduction
Sphingomyelin is one of the components of cell membranes, and its important
metabolites ceramide (Cer), sphingosine (Sp) and sphingosine-1-phosphate (S1P) play
important roles in regulating cell proliferation, migration and apoptosis. Cer and Sp can
inhibit cell growth and promote apoptosis, whereas S1P promotes cell growth and
inhibits apoptosis[1]. Sp can be phosphorylated to S1P by sphingosine kinases (SphKs),
which are important rate-limiting enzymes that regulate the homeostasis of the above
substances in cells[2]. There are two SphKs in mammals, SphK1[3] and SphK2[4].
SphK2 is mainly located in the cell nuclei. Recent studies have found that selective
inhibition of the function of SphK2 can play a role in the treatment of various cancers
such as liver cancer[5] and multiple myeloma[6]. However, the mechanisms for the
physiological and pathological effects of SphK2 are still poorly understood. SphK1 is
mainly located in the cytosol of eukaryotic cells, where it serves as a key kinase in the
SphK1-S1P-S1P receptor (SphK1-S1P-S1PRs) signaling pathway (Fig. 1). By
controlling the level of S1P, Cer and Sp in the cell, this enzyme regulates proliferation
and apoptosis[7,8]. SphK1 is overexpressed in many tumor tissues[9,10] and is thus an
ideal target for the treatment of cancers[11].
OH
HO
C
13H27
NH2
S1P
Sp
S1PRs
Sphingomyelin
Ceramide
Plasma
Membrane
CerS
G protein
Sphingosine
OH
Ceramide
ATP
HO
C13H27
HN C17H35
Cer
O
S1P
SphK1
PI3K
Akt
Ras
Rac
PLC
ADP
S1P
TRAF-2
ERK
NF-
B

OH
O
P
HO
O
C13H27
-
O
NH2
S1P
Survival
Proliferation
Migration
SphK1
Fig. 1 SphK1-S1P-S1PRs signaling pathway

In the past few decades, various types of SphKs inhibitors have been developed[12]
(Fig. 2). N,N-Dimethylsphingosine (DMS)[13] and SKI-II[14] can inhibit SphK1 and
SphK2, protein kinase C and other kinases with low selectivity. PF-543 is a highly
selective SphK1 inhibitor with 100-fold greater selectivity for SphK1 compared with
SphK2; however, it cannot induce apoptosis in cancer cells[15,16]. Natural products
are always an important source of drugs and lead compounds. The active natural
product jaspineꢀB (Fig. 2), which wasꢀisolated from the marine sponges Jaspis sp. in
2003[17], can be considered as an anhydrophytosphingosine derivative. JaspineꢀB
containsꢀanꢀall-cis-2,3,4-trisubstitutedꢀtetrahydrofuranꢀringꢀwithꢀtheꢀ(2S,3S,4S)
absoluteꢀconfigurationꢀandꢀexhibitsꢀpotent antiproliferative activitiesꢀagainstꢀvarious
human cancer cell lines by inhibiting SphKs[18-21]. Its 2-epimer has greater selectivity
for SphK1[22] and demonstrates potent antiproliferative activities. Therefore, 2-epi-
jaspine B can be used as an ideal precursor for the development of selective inhibitors
of SphK1.
OH
O O
S
OH
S
O
HO
C13H27
NH
N
N
N
Cl
HO
1
2
3
PF-543  
DMS  
SKI-II  
H2N
H2N
OH
OH
C14H29
C14H29
O
O
5
2-epi-jaspine B  
4
jaspine B  
Fig. 2 Structures of SphKs inhibitors
Several synthetic[23-26] and structure–activity relationship (SAR) studies[27-29]
have been conducted focused on jaspine B and 2-epi-jaspine B. Our group[30] has
completed the total synthesis of tetrahydropyrrolidine analogs of 2-epi-jaspine B. Based
on preliminary work, we continued to synthesize a series of novel 2-epi-jaspine B
analogs, for which biological evaluation (including kinases and cells) and SAR analyses
were also studied. These studies have contributed to the discovery of highly active and
selective SphK1 inhibitors.

2. Results and discussion
2.1 Design and synthesis
The ligand-binding pocket of SphK1 is a “J-shaped” cavity. The head of the
ligand-binding pocket of SphK1 is hydrophilic, wherein the corresponding amino acid
residues can form hydrogen bonds with SphK1 inhibitors, and the endogenous substrate
Sp can be phosphorylated to S1P. The tail portion of the binding pocket occupies a
major portion of the active cavity and is a hydrophobic cavity that can accommodate a
larger hydrophobic group or the center of an aromatic ring. SphK1 inhibitors regulate
SphK1 function by interacting with the ligand-binding pocket of SphK1. Therefore, the
design of an SphK1 inhibitor must be such that it cannot be phosphorylated and must
fit appropriately into the ligand-binding pocket.
Given the analysis of the SphK1 binding pocket, as shown in Fig. 3, we retained
the hydroxyl and amino groups on the tetrahydrofuran ring of 2-epi-jaspine B that acted
as hydrogen bond donors/receptors and allowed them to form hydrogen bonds with the
hydrophilic portion of the head of the binding pocket. Simultaneously, hydrophobic
groups or aromatic ring centers of various sizes were introduced at the 2-position chiral
side chain to match the tail hydrophobic cavity of the binding pocket, and the effects of
the substituent groups on the biological activity of the analogs were investigated. In this
context, we used the bioisosteric replacement to replace the tetrahydrofuran ring with a
tetrahydropyrrole ring, which retained the rigid five-membered ring, and introduced an
NH group that acted as a hydrogen bond donor/acceptor (forming a salt bridge with the
amino acid residues) and enhanced the affinity of the compound for SphK1.
substituted aromatic ring;
large group
H2N
OH
H2N
OH
hydrophobic group
N
O
H
sosterism
bioi
NH
Fig. 3 Design of tetrahydropyrrolidine analogs
We synthesized a series of novel 2-epi-jaspine B analogs, YHR1-YHR17, based

on our previous studies, as well as the SAR studies of jaspine B and its 2-isomer, 2-epi-
jaspine B. To obtain YHR1-YHR17, we first carried out the synthesis of terminal
olefins shown in Scheme 1.
NaH
Br

O
6
OH
6
DMF, 0 C to r.t.
76
6
7
H1
Scheme 1 Synthesis of H1
As shown in Scheme 1, the synthesis of terminal olefin H1 (76% yield) was
obtained by Williamson condensation of a known alcohol 6 with a halogenated olefin
7. H2-H14 were readily prepared in the same manner in 67%-82% yields, and the
structures of the olefins are listed in Table 1.
Table 1 Structures of H1-H14
Code
Structure
Code
Structure
F
O
7
H1
H8
O
6
Cl
F
F
H2
H3
H4
H9
O
7
O
7
F
F
CF3
H10
H11
O
8
O
7
O
O
7
O
O
CN
H5
H6
H7
H12
H13
H14
O
6
O
7
Cl
F
O
7
O
7
O
8
O
7
O
The pyrrolidine intermediates 8[30] and H14 were subjected to cross metathesis

reaction under the catalysis of the Grubbs2ndꢀcatalyst, followed by hydrogenating the
double bond and deprotecting the protecting groups on the amino and hydroxyl groups
to give YHR17 in 37% yield, as shown in Scheme 2. We used the same procedure as
for YHR17 to synthesize a series of 2-epi-jaspine B analogs, and their structures are
listed in Table 2. All 2-epi-jaspine B analogs were characterized by 1H NMR, 13C NMR
and HRMS spectra.
O
8
O
H14
BnHN
OPMB
(1) Grubbs2nd, CH2Cl2, reflux
H2N
OH
(2) PhSH, CsCO3, MeCN, 50 °C
O
8
N
N
H
O
(3) Pd(OH)2/C, Pd/C, H2, MeOH, r.t.
(4) CAN, MeCN/H2O (4:1), r.t.
Ns
YHR17
8
Scheme 2 Synthesis of YHR17
During the removal of the OPMB protecting group by CAN, there were two
findings: 1) The CAN reagent eliminated the chlorine on the benzene ring, which
resulted in the corresponding compounds YHR9 and YHR11. 2) The CAN reagent
removed the naphthylmethyl group (H13) of the ether group side chain on the
hydrophobic group to form a hydroxyl group, which resulted in YHR16.
H2N
H2N
H2N
H2N
OPMB
OH
Cl
CAN
CAN
O
10
O
10
N
H
N
H
YHR9
OH
H2N
OPMB
F
F
O
10
O
N
H
N
10
H
Cl
YHR11
H2N
OPMB
OH
CAN
O
10
OH
10
N
N
H
H
YHR16
Scheme 3 CAN reagent eliminates chlorine and aromatic ring
Table 2 Structures of YHR1-YHR17

H2N
H2N
H2N
OH
C
OH
C
OH
C
14H29
12H25
16H33
N
N
N
H
H
H
YHR1
YHR2
YHR3
H2N
H2N
H2N
OH
OH
OH
O
9
O
10
O
11
N
N
H
N
H
H
YHR4
YHR5
OH
YHR6
OH
H2N
H2N
H2N
OH
O
10
O
10
O
10
N
N
H
N
H
H
YHR7
YHR8
YHR9
F
F
H2N
H2N
OH
OH
OH
F
F
H2N
OH
F
O
10
O
10
O
10
N
N
H
F
N
H
H
YHR10
YHR11
YHR12
H2N
OH
H2N
CF3
H2N
OH
O
O
CN
O
10
O
10
O
10
N
N
H
N
H
H
YHR13
YHR14
YHR15
H2N
H2N
OH
OH
OH
10
O
11
N
H
N
H
O
YHR16
YHR17
2.2 SAR studies
To carry out detailed structure-activity relationship (SAR) studies, all 2-epi-
jaspine B analogs (YHR1-YHR17) were evaluated for antiproliferative activities on
A549 and LOVO cell lines, and some of them were selected for preliminary tests for
the inhibition of SphKs. The data for these biological evaluations are shown in Table
3 and Table 4. Based on the data in Table 3 and Table 4, we performed SAR analysis
of the 2-epi-jaspine B analogs (YHR1-YHR17), and the results are summarized as
follows.
Table 3 Antiproliferative activities of 2-epi- jaspine B and YHR1-YHR17

IC50 (μM)a
Compounds
A549
LOVO
0.32±0.01
0.05±0
2-epi-jaspine B
YHR1
1.77±0.22
0.02±0
YHR2
0.78±0.06
3.02±0.18
7.68±0.66
0.77±0.07
0.36±0.02
1.68±0.12
1.81±0.16
0.71±0.06
2.88±0.23
1.05±0.08
0.67±0.04
0.54±0.03
9.55±1.80
0.73±0.06
0.95±0.07
5.68±0.55
0.49±0.05
0.14±0.01
4.42±0.29
>10
YHR3
YHR4
YHR5
1.96±0.16
0.18±0.01
1.4±0.11
0.66±0.05
1.57±0.13
2.06±0.19
0.97±0.07
0.58±0.05
1.15±0.10
>10
YHR6
YHR7
YHR8
YHR9
YHR10
YHR11
YHR12
YHR13
YHR14
YHR15
YHR16
YHR17
Cisplatinb
0.19±0.01
4.42±0.39
0.68±0.05
0.72±0.06
aIC50 values were calculated from three independent experiments using the MTT assay
b
after a 48 h treatment. The values are reported as the Means ± SD. Cisplatin is a
positive control anticancer drug. A549: Human non-small cell lung cancer cell line;
LOVO: Human colon cancer cell line.
The results of Table 3 and Table 4 show that the antiproliferative activities of the
modified tetrahydropyrrolidine analogs are close to or better than those of 2-epi-jaspine
B. The replacement of the tetrahydrofuran ring in 2-epi-jaspine B by a tetrahydropyrrole

ring to obtain YHR1 resulted in dramatic increases in the antiproliferative activities
against a human non-small cell lung cancer cell line (A549) (IC50 = 20 nM) and a human
colon cancer cell line (LOVO) (IC50 = 50 nM). However, as the carbon chain of the
aliphatic chain increased or decreased, the antiproliferative activity decreased
significantly, and the effect of an increase in the number of carbon atoms is particularly
obvious (YHR3). To increase the matching degree of the compound to SphK1, we
introduced a hydrophobic group or an aromatic ring of various sizes at the end of the
long chain to investigate the effects of these substitutions on the selectivity and
antiproliferative activity. The introduction of the terminal cyclohexane group (YHR5
and YHR6) lead to a 3 or 4-fold increase in the antiproliferative activity compared with
2-epi-jaspine B. These effects on the activities were affected by the length of the carbon
chain, and the activities increased as the carbon chain length increased. The
replacement of the cyclohexane group in YHR4 by a benzene (YHR8) and the
cyclohexane group in YHR5 by an isobutyl group (YHR7) lead to different results: the
isobutyl group reduced the antiproliferative activity whereas benzene increased it. As
shown in Table 2, the introduction of a large hydrophobic group increased the
selectivity of the derivative for SphK1 compared to the long chain and 2-epi-jaspine B,
which suggested that the large hydrophobic group was important for selectivity and
antiproliferative activity. We next examined the effects of the α carbon on right side
chain of the ether bond on antiproliferative activities by YHR9-YHR11. When the
methyl group was introduced and there was an electron-withdrawing substituent on the
benzene ring (YHR10), the antiproliferative activity was less than that of YHR9, which
lacked these groups. Based on the results of this analysis, we concentrated on the
favorable structural optimization on YHR17, hoping to increase the selectivity or
improve the potency. To our surprise, as shown in Table 3 and Table 4, the selectivity
of YHR17 for SphK1 over SphK2 was more than 125-fold, and this compound had
potent antiproliferative activity against LOVO (IC50 = 0.68 μM). We introduced various
electron-withdrawing and electron-donating substituents on the benzene to synthesize
the analogs YHR12-YHR15. The results suggested that the electron-withdrawing
substituents on the benzene ring could enhance the antiproliferative activity of the

analogs on the test cell lines whereas electron-donating substituents had the opposite
effect. The unexpectedly obtained analog YHR16 also exerted good antiproliferative
activity, which indicated that the hydroxyl group of the long chain tail also played a
certain role. Compared with 2-epi-jaspine B, YHR17 had better selectivity, and the
antiproliferative activity was also maintained. Thus, we selected two additional cancer
cell lines to investigate the biological effects of YHR17.
Table 4 Comparison of 2-epi-jaspine B and its analogs on the inhibition of SphKs.
IC50 (μM)a
SphK1
Selectivity
(SphK2/SphK1)
>7.3
Compounds
SphK2
>100
YHR3
YHR4
13.8 ± 0.7
9.7 ± 0.3
4.9 ± 0.3
6.9 ± 0.7
0.8 ± 0.1
4.3 ± 0.4
>100
>10.3
YHR5
>100
>20.4
YHR7
>100
>14.5
YHR17
>100
>125
5.8
2-epi-jaspine B
24.8 ± 2.8
aIC50 values were calculated from three independent experiments. The values were
reported as the Means ± SD.
Next, we examined the antiproliferative activities of 2-epi-jaspine B and YHR17
on a human malignant melanoma cell line (A375) and a human hepatoma cell line
(HepG2). In addition, a normal human cardiac fibroblast cell line (HCFB) was also
examined, with the aim of assessing the toxicity of YHR17. The results in Table 5
shown that YHR17 displayed potent antiproliferative activities on A375 (IC50 = 0.7 μM)
and HepG2 (IC50 = 1.46 μM) cells. Furthermore, YHR17 has no obvious cytotoxicity
in the normal cell line HCFB (IC50 >100 μM).
Table 5 Comparison of 2-epi-jaspine B and YHR17 on the growth of various cell lines.
IC50 (μM)a
compounds
A375
HepG2
HCFB
>100
2-epi-jaspine B
0.53±0.01
2.12±0.15

YHR17
0.70±0.09
1.3±0.11
1.46±0.12
1.21±0.16
>100
>100
Cisplatinb
aIC50 values are reported as the Means ± SD, which were derived from three
independent experiments. bCisplatin is a positive control anticancer drug. A375: Human
malignant melanoma cell line; HepG2: Human hepatoma cell line; HCFB: Human
cardiac fibroblast cell line.
2.3 YHR17 arrested the cell cycle at G1
The data in Table 5 shown that YHR17 has a significant antiproliferative activity
on the A375 cell line. We therefore continued to study its biological effects in the A375
cell line. A propidium iodide (PI) staining kit was used to investigate the effect of
YHR17 on the cell cycle distribution. A375 cells were stained with PI and analyzed on
a flow cytometer after treatment with YHR17 (0.1 0.3, 1, 3 μM) and the control for 36
h.
Fig. 4 YHR17 induced cell cycle arrest in A375 cells. The cells were treated with
YHR17 (0.1, 0.3, 1, 3 μM) for 36ꢀh, fixed in cold 70% ethanol at -20ꢀ°C, and then
stained with propidium iodide dye (10ꢀµg/mL). The cell cycle analysis was evaluated
using flow cytometry (FACSCalibur, Becton Dickinson, USA). The insert bar graph

showed the percentage (%) of the cells in the G1 and G2 phases.
Fig. 4 shows that as the concentration of YHR17 increased, the cells in the G1
phase accumulated significantly compared to the control group (63.94%), with a range
from 65.65% (0.1 μM) to 82.59% (3 μM), and exhibited concentration-dependence.
Meanwhile, the cells in the G2 and S phases decreased from 13.28% to 0 and from
22.78% to 17.14%, respectively. From the above results, the mechanism of cytotoxicity
of YHR17 may be that it can arrest cell division at G1 phase.
2.4 YHR17 induced A375 cell apoptosis
A375 cells were examined using the Annexin V-FITC/PI FACS assay after being
treated with YHR17 (0.1, 0.3, 1, 3, 10, 33 μM) and Cisplatin (10 μM) for 48 h, with
the aim of exploring the mechanism of cell death. Fig. 5 shows that as the concentration
of YHR17 increased, the percentage of the apoptotic population in A375 cells increased
significantly, with a range of 4.81% to 71.17% at 48 h. The apoptotic rate of A375 cells
were positively correlated with the concentration of YHR17 in a dose-dependent
manner.

Fig. 5 YHR17 induced apoptosis in A375 cells. (A) A375 cells were treated for 48 h
with various concentrations of YHR17 as indicated. The cells were then stained with
annexin V/PI, and apoptosis was analyzed using flow cytometry. The insert bar graph
shows the statistics of total apoptotic cell percentages from duplicate experiments. **P
< 0.05, *P < 0.01, versus the control group.
2.5 Docking mode analysis
To further investigate the reasons for the high selectivity of YHR17 and support
the SAR studies, we analyzed the molecular docking of YHR17 with SphK1. The
molecular docking analysis was performed using Discovery Studio 2019 based on the
cocrystal structure of SphK1 and sphingosine (PDB ID: 3VZB).
As shown in Fig. 6A, the amino and hydroxyl groups on the tetrahydrofuran ring
of 2-epi-jaspine B can form hydrogen bounds with the amino acid residues LEU268
and SER168 in the ligand-binding pocket of SphK1, and the long-chain aliphatic
hydrocarbon of the 2-position can also contribute a certain hydrophobic effect. At the
same time, it can be seen from the combination pattern that there is still a large space
at the end of the 2-position, which could theoretically accommodate larger hydrophobic
groups. As shown in Fig. 6B, the amino and hydroxyl groups on the ring of cationic
form of YHR17 can form hydrogen bonds with LEU268, SER168 and ASP81, and the
+
NH2 on the pyrrole ring can also serve as a hydrogen bond donor to ASP178 whereas
the tetrahydrofuran ring of 2-epi-jaspine B has no such effect. In addition, a large group
in the YHR17 tail has a better hydrophobic effect than 2-epi-jaspine B. We then

performed a molecular docking analysis of the endogenous substrate sphingosine of
SphK1 with YHR17. As shown in Fig. 6C, the spatial orientation of YHR17 (thick
gray line) is highly consistent with the substrate sphingosine (thin yellow line). From
the above docking models, it can be concluded that the reasons for the relatively high
inhibitory activity of YHR17 can be attributed to three points. 1) YHR17 forms more
hydrogen bounds with the ligand-binding pocket of SphK1. 2) The tetrahydropyran ring
at the end of the side chain acts as a hydrophobic center that can interact
hydrophobically with the corresponding amino acid residues. 3) The structure of
YHR17 is highly matched to the hydrophobic pocket of SphK1. The above analyses
provide a reasonable explanation for the inhibitory activities and high selectivity of
YHR17. Furthermore, the analyses provide a foundation for further studies, in which
different hydrophobic groups as alkyl side chains may improve the isoform selectivity
and inhibitory activity of these tetrahydropyrrolidine analogs.
A

B
C
Fig. 6 (A) Electrostatic interactions between 2-epi-jaspine B and SphK1. Amino and
hydroxyl groups are blue and red, respectively. (B) Predicted binding mode of YHR17
and electrostatic interactions with SphK1. Amino and hydroxyl groups are blue and red,
respectively. (C) Predicted binding mode of YHR17 and sphingosine. The thick gray
line represents YHR17, and the thin yellow line represents sphingosine.
3. Conclusion
In this study, we designed and synthesized some new analogs of 2-epi-jaspine B
and tested their cytotoxic activities against A549, LOVO, A375, HepG2 cell lines and
a normal cell line HCFB. In addition, we tested the inhibitory activities of these analogs
against SphK1 and SphK2. Based on the results of the above experiments, we identified
a novel 2-epi-jaspine B analog YHR17, which exhibited potent antiproliferative
activities with IC50 values of 0.68 and 0.7 μM against LOVO and A375, respectively.
Moreover, the IC50 value of YHR17 against the normal cell line HCFB is more than
100 μM, thus demonstrating no obvious cytotoxicity. YHR17 exhibited an anticancer
activity comparable to that of 2-epi-jaspine B and exhibited inhibition of SphK1 with
more than 125-fold selectivity over SphK2. Further biological evaluations suggested
that YHR17 arrested the cell cycle at the G1 phase and induced A375 cell apoptosis in
a dose-dependent manner. We performed a systematic SAR analysis of 2-epi-jaspineB
analogs and verified the results by molecular docking, based on the above data. All the
results confirmed that YHR17 is a potent and selective SphK1 inhibitor with good
development prospects.

4. Experimental section
4.1 Materials and methods
All chemicals were purchased from commercial sources and used as received. For
detailed information on the materials and methods, please see Supporting Information.
4.2 Synthesis of intermediates
All the synthetic procedures of intermediates are listed in Supporting Information.
4.3 Chemical synthesis and spectra data of the long-chain terminal olefins H1-H14
((oct-7-en-1-yloxy)methyl)cyclohexane (H1)
To a stirred solution of cyclohexylmethanol (6) (492 μL, 4.0 mmol, 2 eq) in DMF
(7 mL) under argon was added NaH (60% suspension in mineral oil; 160 mg, 4.0 mmol,
2 eq) at 0 °C. The suspension was stirred at 0 °C for 30 min, then 8-bromodec-1-octene
(7) (336 μL, 2.0 mmol, 1 eq) was added dropwise to the mixture. The mixture was
allowed to warm to room temperature and stirred for 1 h. After the reaction was
quenched with saturated NH4Cl, the whole was extracted with Et2O. The extract was
washed with H2O and brine, then dried over MgSO4. The filtrate was concentrated
under reduced pressure to give an oily residue, which was purified using silica gel
chromatography (petroleum ether:EtOAc = 40:1) to afford H1 (341 mg, 76% yield) as
a colorless oil. 1H NMR (CD3OD, 600 MHz) δ (ppm): 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz,
1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H), 4.93 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 3.38 (t, J =
6.7 Hz, 2H), 3.19 (d, J = 6.6 Hz, 2H), 2.01–2.07 (m, 2H), 1.64–1.77 (m, 5H), 1.52–1.61
13
(m, 3H), 1.12–1.42 (m, 9H), 0.91 (dq, J = 12.3, 3.2 Hz, 2H). C NMR (CDCl3, 150
MHz) δ (ppm): 139.12, 114.16, 76.85, 71.09, 38.08, 33.76, 30.20 (2C), 29.71, 28.99,
28.89, 26.70, 26.07, 25.92 (2C). HRMS (ESI): calcd for C15H29OH+ [M+H]+ 225.2218,
found 225.2220.
((non-8-en-1-yloxy)methyl)cyclohexane (H2)
Using a procedure with cyclohexylmethanol (6) (492 μL, 4.0 mmol, 2 eq) similar
to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0 mmol, 1 eq)
was converted to H2 (391 mg, 82% yield) as a colorless oil. 1H NMR (CDCl3, 600 MHz)
δ (ppm): 5.80 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H), 4.92
(ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 3.37 (t, J = 6.7 Hz, 2H), 3.19 (d, J = 6.6 Hz, 2H), 2.06

– 2.01 (m, 2H), 1.77–1.65 (m, 5H), 1.60–1.53 (m, 3H), 1.38–1.15 (m, 11H), 0.91 (qd,
13
J = 12.3, 3.1 Hz, 2H). C NMR (CDCl3, 150 MHz) δ (ppm): 139.16, 114.12, 76.85,
71.11, 38.07, 33.79, 30.19 (2C), 29.74, 29.34, 29.09, 28.88, 26.69, 26.14, 25.91 (2C).
HRMS (ESI): calcd for C16H30OH+ [M+H]+ 239.2375, found 239.2378.
((dec-9-en-1-yloxy)methyl)cyclohexane (H3)
Using a procedure with cyclohexylmethanol (6) (492 μL, 4.0 mmol, 2 eq) similar
to that described for preparation of H1, 10-bromodec-1-decene (401 μL, 2.0 mmol, 1
eq) was converted to H3 (414 mg, 82% yield) as a colorless oil. 1H NMR (CDCl3, 600
MHz) δ (ppm): 5.81 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H),
4.92 (ddt, J = 10.2, 2.4, 1.3 Hz, 1H), 3.37 (t, J = 6.7 Hz, 2H), 3.19 (d, J = 6.6 Hz, 2H),
2.01–2.06 (m, 2H), 1.64–1.77 (m, 5H), 1.51–1.61 (m, 3H), 1.15–1.37 (m, 13H), 0.91
(qd, J = 12.3, 3.1 Hz, 2H). 13C NMR (CDCl3, 150 MHz) δ (ppm): 139.20, 114.11, 76.86,
71.14, 38.08, 33.82, 30.20 (2C), 29.76, 29.46 (2C), 29.10, 28.94, 26.71, 26.19, 25.92
(2C). HRMS (ESI): calcd for C17H32OH+ [M+H]+ 253.2531, found 253.2517.
9-isobutoxynon-1-ene (H4)
Using a procedure with isobutyl alcohol (368 μL, 4.0 mmol, 2 eq) similar to that
described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0 mmol, 1 eq) was
converted to H4 (301 mg, 76% yield) as a colorless oil. 1H NMR (CDCl3, 400 MHz) δ
(ppm): 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.03-4.90 (m, 2H), 3.39 (t, J = 6.7 Hz,
2H), 3.16 (d, J = 6.7 Hz, 2H), 2.04 (q, J = 7.1 Hz, 2H), 1.80–1.90 (m, 1H), 1.53–1.60
(m, 2H), 1.25–1.42 (m, 8H), 0.90 (d, J = 6.7 Hz, 6H). 13C NMR (CDCl3, 100 MHz) δ
(ppm): 139.19, 114.12, 77.85, 71.06, 33.79, 29.74, 29.34, 29.09, 28.88, 28.44, 26.15,
19.43 (2C). HRMS (ESI): calcd for C13H26OH+ [M+H]+ 199.2062, found 199.2056.
((oct-7-en-1-yloxy)methyl)benzene (H5)
Using a procedure with benzyl alcohol (416 μL, 4.0 mmol, 2 eq) similar to that
described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0 mmol, 1 eq) was
converted to H5 (323 mg, 74% yield) as a colorless oil. 1H NMR (CDCl3, 600 MHz) δ
(ppm): 7.34 (d, J = 4.6 Hz, 4H), 7.27 (ddd, J = 8.8, 5.0, 3.9 Hz, 1H), 5.80 (ddt, J = 16.9,
10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H), 4.93 (ddt, J = 10.2, 2.3, 1.2 Hz, 1H),
4.50 (s, 2H), 3.46 (t, J = 6.6 Hz, 2H), 2.02–2.06 (m, 2H), 1.56–1.65 (m, 2H), 1.30–1.41

13
(m, 6H). C NMR (CDCl3, 150 MHz) δ (ppm): 139.13, 138.72, 128.35 (2C), 127.62
(2C), 127.47, 114.20, 72.88, 70.48, 33.75, 29.74, 28.97, 28.88, 26.08. HRMS (ESI):
calcd for C15H22ONa + [M+Na]+ 241.1568, found 241.1563.
1-chloro-4-(1-(non-8-en-1-yloxy)ethyl)benzene (H6)
Using a procedure with 1-(4-chlorophenyl) ethanol (541 μL, 4.0 mmol, 2 eq)
similar to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0
1
mmol, 1 eq) was converted to H6 (410 mg, 73% yield) as a colorless oil. H NMR
(CDCl3, 600 MHz) δ (ppm): 7.29–7.32 (m, 2H), 7.22–7.25 (m, 2H), 5.80 (ddt, J = 16.9,
10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H), 4.93 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H),
4.35 (q, J = 6.5 Hz, 1H), 3.23–3.29 (m, 2H), 2.00–2.06 (m, 2H), 1.53–1.57 (m, 2H),
13
1.40 (d, J = 6.5 Hz, 3H), 1.34–1.38 (m, 2H), 1.24–1.31 (m, 6H). C NMR (CD3OD,
150 MHz) δ (ppm): 142.82,138.70, 132.67, 128.14 (2C), 127.41 (2C), 113.39, 77.18,
68.41, 33.49, 29.50, 28.94, 28.76, 28.67, 25.82, 22.97. HRMS (ESI): calcd for
C17H25ClOH+ [M+H]+ 281.1672, found 281.1655.
1-fluoro-4-(1-(non-8-en-1-yloxy)ethyl)benzene (H7)
Using a procedure with 1-(4-fluorophenyl) ethanol (499 μL, 4.0 mmol, 2 eq)
similar to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0
1
mmol, 1 eq) was converted to H7 (434 mg, 82% yield) as a colorless oil. H NMR
(CDCl3, 600 MHz) δ (ppm): 7.27 (ddd, J = 8.3, 5.2, 2.8 Hz, 2H), 7.05- 6.99 (m, 2H),
5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.98 (dq, J = 17.1, 1.6 Hz, 1H), 4.92 (ddt, J =
10.2, 2.2, 1.2 Hz, 1H), 4.35 (q, J = 6.5 Hz, 1H), 3.22–3.29 (m, 2H), 2.00–2.06 (m, 2H),
1.52–1.57 (m, 2H), 1.40 (d, J = 6.5 Hz, 3H), 1.33–1.39 (m, 2H), 1.26–1.30 (m, 6H). 13C
NMR (CD3OD, 150 MHz) δ (ppm): 162.14 (d, J = 241.5 Hz, 1C), 139.94, 138.69,
127.62, 127.57, 114.73, 114.58, 113.37, 77.18, 68.27, 33.48, 29.49, 28.94, 28.75, 28.67,
25.83, 23.04. HRMS (ESI): calcd for C17H25FOH+ [M+H]+ 265.1968, found 265.1952.
2-chloro-4-fluoro-1-((non-8-en-1-yloxy)methyl)benzene (H8)
Using a procedure with 2-chloro-4-fluorobenzyl alcohol (478 μL, 4.0 mmol, 2 eq)
similar to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0
1
mmol, 1 eq) was converted to H8 (444 mg, 78% yield) as a colorless oil. H NMR
(CDCl3, 600 MHz) δ (ppm): 7.45 (dd, J = 8.6, 6.2 Hz, 1H), 7.10 (dd, J = 8.5, 2.6 Hz,

1H), 6.98 (td, J = 8.4, 2.6 Hz, 1H), 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.99 (dq, J =
17.1, 1.6 Hz, 1H), 4.93 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 4.53 (s, 2H), 3.52 (t, J = 6.6 Hz,
2H), 2.01–2.07 (m, 2H), 1.60–1.67 (m, 2H), 1.35–1.41 (m, 4H), 1.31 (dt, J = 7.3, 3.4
Hz, 4H). 13C NMR (CDCl3,150 MHz) δ (ppm): 161.77 (d, J = 247.5Hz, 1C), 139.14,
133.42 (d, J = 10.5Hz, 1C), 132.40 (d, J = 4.5Hz, 1C), 130.04 (d, J = 4.5Hz, 1C), 116.58
(d, J = 24.0Hz, 1C), 114.18, 113.88 (d, J = 19.5Hz, 1C), 71.05, 69.26, 33.79, 29.71,
29.30, 29.07, 28.87, 26.14. HRMS (ESI): calcd for C16H22ClFOH+ [M+H]+ 285.1421,
found 285.1420.
1,2,4,5-tetrafluoro-3-methyl-6-((non-8-en-1-yloxy)methyl)benzene (H9)
Using a procedure with 2,3,5,6-tetrafluoro-4-methylbenzyl alcohol (586 μL, 4.0
mmol, 2 eq) similar to that described for preparation of H1, 9-bromodec-1-nonene (371
1
μL, 2.0 mmol, 1 eq) was converted to H9 (509 mg, 80% yield) as a colorless oil. H
NMR (CDCl3, 600 MHz) δ (ppm): 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.98 (dq, J =
17.1, 1.6 Hz, 1H), 4.92 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 4.58 (s, 2H), 3.47 (t, J = 6.6 Hz,
2H), 2.27 (t, J = 2.1 Hz, 3H), 2.00–2.05 (m, 2H), 1.57 (p, J = 6.7 Hz, 2H), 1.31–1.39
(m, 4H), 1.26–1.30 (m, 4H). 13C NMR (CDCl3, 150 MHz) δ (ppm): 145.89 (ddd, J =
14.3, 6.7, 3.9 Hz, 1C), 145.63 (ddd, J = 14.4, 6.7, 4.0 Hz, 1C), 144.25 (ddd, J = 14.3,
6.7, 3.9 Hz, 1C), 144.01 (ddd, J = 14.4, 6.8, 3.9 Hz, 1C), 139.18, 116.29 (t, J = 19.1
Hz, 1C), 114.15, 113.66 (t, J = 18.0 Hz, 1C), 70.90, 59.78, 33.78, 29.56, 29.24, 29.05,
28.86, 25.98, 7.63. HRMS (ESI): calcd for C17H22F4OH+ [M+H]+ 319.1685, found
319.1677.
1-((non-8-en-1-yloxy)methyl)-4-(trifluoromethyl)benzene (H10)
Using a procedure with 4-(trifluoromethyl) benzyl alcohol (548 μL, 4.0 mmol, 2
eq) similar to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0
1
mmol, 1 eq) was converted to H10 (457 mg, 76% yield) as a colorless oil. H NMR
(CDCl3, 600 MHz) δ (ppm): 7.60 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 5.81
(ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H), 4.91–4.95 (m, 1H),
4.55 (s, 2H), 3.48 (t, J = 6.6 Hz, 2H), 2.04 (q, J = 7.0 Hz, 2H), 1.63 (dt, J = 14.6, 6.7
Hz, 2H), 1.37 (q, J = 11.7, 9.1 Hz, 4H), 1.31 (dq, J = 7.2, 4.4, 3.3 Hz, 4H). 13C NMR
(CDCl3, 150 MHz) δ (ppm): 142.90, 139.15, 129.65 (q, J = 32.3 Hz, 1C), 127.48 (2C),

125.29 (q, J = 3.8 Hz, 2C), 123.32, 114.19, 72.06, 70.91, 33.79, 29.73, 29.32, 29.07,
28.87, 26.15. HRMS (ESI): calcd for C17H23F3OH+ [M+H]+ 301.1779, found 301.1775.
2,4-dimethoxy-1-((non-8-en-1-yloxy)methyl)benzene (H11)
Using a procedure with 2,4-dimethoxybenzyl alcohol (606 μL, 4.0 mmol, 2 eq)
similar to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0
1
mmol, 1 eq) was converted to H11 (456 mg, 78% yield) as a colorless oil. H NMR
(CDCl3, 600 MHz) δ (ppm): 7.24 (d, J = 8.2 Hz, 1H), 6.43–6.49 (m, 2H), 5.80 (ddt, J
= 17.0, 10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.7 Hz, 1H), 4.92 (ddt, J = 10.2, 2.3, 1.2
Hz, 1H), 4.46 (s, 2H), 3.80 (d, J = 1.6 Hz, 6H), 3.46 (t, J = 6.7 Hz, 2H), 2.00–2.07 (m,
13
2H), 1.56–1.64 (m, 2H), 1.28–1.39 (m, 8H). C NMR (CDCl3,150 MHz) δ (ppm):
160.43, 158.39, 139.19, 130.10, 119.53, 114.12, 103.89, 98.42, 70.35, 67.30, 55.39,
55.35, 33.79, 29.76, 29.33, 29.10, 28.88, 26.16. HRMS (ESI): calcd for C18H28O3H+
[M+H]+ 293.2117, found 293.2100.
4-((non-8-en-1-yloxy)methyl)benzonitrile (H12)
Using a procedure with 4-(hydroxymethyl) benzonitrile (459 μL, 4.0 mmol, 2 eq)
similar to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0
1
mmol, 1 eq) was converted to H12 (422 mg, 82% yield) as a colorless oil. H NMR
(CDCl3, 400 MHz) δ (ppm): 7.63 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 5.81
(ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.96 (dd, J = 23.0, 13.7 Hz, 2H), 4.55 (s, 2H), 3.49 (t,
J = 6.6 Hz, 2H), 2.04 (q, J = 7.1 Hz, 2H), 1.62 (td, J = 13.5, 6.7 Hz, 2H), 1.28–1.43 (m,
8H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 144.39, 139.11, 132.19 (2C), 127.64 (2C),
118.91, 114.20, 111.16, 71.87, 71.12, 33.76, 29.69, 29.29, 29.04, 28.85, 26.12. HRMS
(ESI): calcd for C17H23NOH+ [M+H]+ 258.1858, found 258.1860.
2-((non-8-en-1-yloxy)methyl)naphthalene (H13)
Using a procedure with 1-naphthalenemethanol (550 μL, 4.0 mmol, 2 eq) similar
to that described for preparation of H1, 9-bromodec-1-nonene (371 μL, 2.0 mmol, 1 eq)
1
was converted to H13 (378 mg, 67% yield) as a colorless oil. H NMR (CDCl3, 600
MHz) δ (ppm): 8.11 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.2 Hz,
1H), 7.45–7.54 (m, 3H), 7.39–7.44 (m, 1H), 5.79 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.98
(dd, J = 17.1, 1.9 Hz, 1H), 4.91–4.94 (m, 3H), 3.53 (t, J = 6.6 Hz, 2H), 2.01 (q, J = 7.0

Hz, 2H), 1.62 (dt, J = 14.5, 6.7 Hz, 2H), 1.30–1.40 (m, 4H), 1.27 (dt, J = 7.1, 3.4 Hz,
13
4H). C NMR (CDCl3, 150 MHz) δ (ppm): 139.15, 134.10, 133.74, 131.76, 128.46,
128.43, 126.22, 126.04, 125.68, 125.17, 124.07, 114.13, 71.41, 70.54, 33.77, 29.77,
29.26, 29.05, 28.84, 26.15. HRMS (ESI): calcd for C20H26OH+ [M+H]+ 283.2062,
found 283.2072.
4-(dec-9-en-1-yloxy)tetrahydro-2H-pyran (H14)
Using a procedure with tetrahydro-4-pyranol (382 μL, 4.0 mmol, 2 eq) similar to
that described for preparation of H1, 10-bromodec-1-decene (401 μL, 2.0 mmol, 1 eq)
1
was converted to H14 (346 mg, 72% yield) as a colorless oil. H NMR (CDCl3, 600
MHz) δ: 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.99 (dq, J = 17.1, 1.6 Hz, 1H), 4.93
(ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 3.94 (dt, J = 11.7, 4.3 Hz, 2H), 3.48–3.40 (m, 5H),
2.06–2.01 (m, 2H), 1.89 (ddt, J = 11.2, 5.2, 2.7 Hz, 2H), 1.61–1.54 (m, 4H), 1.41–1.27
(m, 10H).13C NMR (CDCl3, 150 MHz) δ: 139.20, 114.11,73.82, 67.78, 65.84 (2C),
33.79, 32.55 (2C), 30.07 29.43, 29.42, 29.07, 28.90, 26.21. HRMS (ESI): calcd for
C15H28O2H+ (M+H)+ 241.2168, found 241.2153.
4.4 Chemical synthesis and spectral data for the tetrahydropyrrolidine analogs
YHR1-YHR17
(2R,3S,4S)-4-amino-2-(11-((tetrahydro-2H-pyran-4-yl)oxy)undecyl)pyrrolidin-3-ol
(YHR17)
4-(dec-9-en-1-yloxy)tetrahydro-2H-pyran (H14) (179 mg, 0.744 mmol, 4 equiv)
and Grubbs2nd catalyst (31 mg, 0.037 mmol, 0.2 eq) were added to a solution of 8 (100
mg, 0.186 mmol, 1 eq) in anhydrousꢀCH2Cl2 (6 mL) under a nitrogen atmosphere.
Afterꢀheatingꢀunderꢀrefluxꢀwithꢀstirringꢀforꢀ7ꢀh,ꢀtheꢀresultingꢀmixtureꢀwasꢀcooledꢀto
roomꢀtemperature and filtered through a short silica gel column, which was eluted with
EtOAc:CH2Cl2:hexane = 3ꢀ:ꢀ2ꢀ:ꢀ5. The solvent was removed under reduced pressure to
give the crude metathesis product as a residue, which was dissolved in anhydrous
MeCN (6 mL). CsCO3ꢀ(52ꢀmg,ꢀ0.372ꢀmmol, 2 eq)ꢀandꢀPhSHꢀ(28.7ꢀL,ꢀ0.28 mmol, 1.2
eq) were added to thisꢀmixture,ꢀandꢀtheꢀresulting mixtureꢀwasꢀstirredꢀatꢀ50 ℃ꢀunder a
hydrogenꢀatmosphere for 3 h. Theꢀresultingꢀmixtureꢀwasꢀcooledꢀtoꢀroomꢀtemperature
andꢀfiltered through a short silica gel column, which was eluted with CH2Cl2. The

solvent was removed under reduced pressure to give the crude metathesis product as a
residue, which was dissolved in anhydrous MeOH (6 mL). The solution was treated
with 20%ꢀPd(OH)2/C (30ꢀmg)ꢀandꢀ10%ꢀPd/C (10 mgꢀ) atꢀroom temperatureꢀunderꢀa
hydrogen ꢀatmosphere and stirredꢀfor 1d. The mixture was then filtered through a pad
of celite and concentrated under reduced pressure to give the crude metathesis product
as a residue, which was dissolved in aꢀmixtureꢀofꢀMeCNꢀandꢀwaterꢀ(4ꢀ:ꢀ1,ꢀ3.7ꢀmL).
Ammonium nitrate (467 mg, 1.78 mmol) was then added to this
solution.ꢀAfterꢀstirringꢀat room temperature for 3 h, the mixture was then concentrated
under reduced pressure to give an oily residue, which was purified by silica gel column
chromatography (MeOH:EtOH:CH2Cl2:NH4OH = 6:12:77:5) to afford YHR17 (24 mg,
[α]2D5
37% yield in 4 steps) as a white solid.
+13 (c = 0.1, MeOH), 1H NMR (CD3OD,
600 MHz) δ (ppm): 3.89 (dt, J = 11.5, 4.3 Hz, 2H), 3.58 (t, J = 5.4 Hz, 1H), 3.53–3.41
(m, 5H), 3.18–3.09 (m, 2H), 2.84 (q, J = 5.9 Hz, 1H), 2.55 (dd, J = 10.6, 7.2 Hz, 1H),
1.92–1.86 (m, 2H), 1.62–1.24 (m, 24H). 13C NMR (CD3OD,150 MHz) δ (ppm): 76.15,
73.63, 67.43, 65.31 (2C), 64.58, 53.54, 50.98, 33.90, 32.23 (2C), 29.72, 29.45, 29.34,
29.31 (2C), 29.29, 29.18, 26.68, 25.94.IR (KBr, cm-1): 3350, 3244, 2922, 2661, 2536,
1641, 1585, 1469, 1361, 1166, 1136, 1114, 1087, 1006, 954, 893, 867, 821, 719, 626,
567. HRMS (ESI): calcd for C14H30N2O2H+ [M+H]+ 357.3117, found 357.3000.
(2R,3S,4S)-4-amino-2-tetradecylpyrrolidin-3-ol (YHR1)
With a four-step conversion using 1-tridecene similar to that described for
preparation of YHR17, 8 was converted to YHR1 (14 mg, 25% yield in 4 steps) as a
[α]2D5
white solid.
+10.4(c = 0.1, MeOH), 1H NMR (CDCl3, 400 MHz) δ (ppm): 3.48
(t, J = 5.6 Hz, 1H), 3.29–3.43 (m, 2H), 2.82 (q, J = 5.6 Hz, 1H), 2.60 (dq, J = 11.6, 6.0,
5.5 Hz, 1H), 1.84 (brs, 4H), 1.54–1.63 (m, 1H), 1.25 (s, 25H), 0.88 (t, J = 6.7 Hz, 3H).
13C NMR (CDCl3, 100 MHz) δ (ppm): 75.72, 65.00, 53.15, 51.73, 34.02, 31.85, 29.55,
29.58 (2C), 29.62 (4C), 29.48, 29.29, 26.75, 22.61,14.00. IR (KBr, cm-1): 3348, 3242,
2918, 2850, 1558, 1469, 1458, 1377, 1350, 1118, 1080, 1028, 956, 908, 894, 869, 825,
719. HRMS (ESI): calcd for C18H38N2OH+ [M+H]+ 299.3062, found 299.3047.
(2R,3S,4S)-4-amino-2-dodecylpyrrolidin-3-ol (YHR2)
With a four-step conversion using 1-undecene similar to that described for

preparation of YHR17, 8 was converted to YHR2 (22 mg, 36% yield in 4 steps) as a
[α]2D5
1
white solid.
+13.3 (c = 0.12, MeOH), H NMR (CD3OD, 600 MHz) δ (ppm):
3.62–3.66 (m, 1H), 3.22 (q, J = 6.7 Hz, 1H), 3.17 (dd, J = 11.0, 6.7 Hz, 1H), 2.90 (q, J
= 5.8 Hz, 1H), 2.62 (dd, J = 11.0, 7.4 Hz, 1H), 1.58–1.65 (m, 1H), 1.24–1.51 (m, 21H),
0.92 (t, J = 7.0 Hz, 3H). 13C NMR (CD3OD, 150 MHz) δ (ppm): 75.93, 64.58, 53.38,
50.68, 33.65, 31.69, 29.44, 29.42, 29.40, 29.38, 29.36, 39.30, 29.10, 26.66, 22.36, 13.07.
IR (KBr, cm-1): 3350, 3244, 2920, 2850, 2667, 2546, 1641, 1591, 1467, 1377, 1352,
1311, 1116, 1080, 1029, 954, 906, 871, 823, 719 cm-1; HRMS (ESI): calcd for
C16H34N2OH+ [M+H]+ 271.2749, found 271.2629.
(2R,3S,4S)-4-amino-2-hexadecylpyrrolidin-3-ol (YHR3)
With a four-step conversion using 1-pentadecene similar to that described for
preparation of YHR17, 8 was converted to YHR3 (22 mg, 36% yield in 4 steps) as a
[α]2D5
1
white solid.
+12.1 (c = 0.12, MeOH), H NMR (CD3OD, 600 MHz) δ (ppm):
3.55–3.58 (m, 1H), 3.15 (q, J = 6.7 Hz, 1H), 3.09 (dd, J = 11.2, 6.7 Hz, 1H), 2.84 (q, J
= 6.3 Hz, 1H), 2.56 (dd, J = 11.2, 7.5 Hz, 1H), 1.48–1.55 (m, 1H), 1.11–1.40 (m, 29H),
13
0.80 (t, J = 7.0 Hz, 3H). C NMR (CDCl3, 150 MHz) δ (ppm): 75.66, 65.09, 53.01,
51.91, 33.98, 31.90, 29.59, 29.63 (2C), 29.67 (6C), 29.52, 29.33, 26.79, 22.66, 14.07.
IR (KBr, cm-1): 3350, 3240, 2956, 2918, 2850, 1558, 1469, 1458, 1261, 1101, 1083,
1028, 954, 914, 883, 852, 804, 765, 719. HRMS (ESI): calcd for C20H42N2OH+ [M+H]+
327.3375, found 327.3231.
(2R,3S,4S)-4-amino-2-(9-(cyclohexylmethoxy)nonyl)pyrrolidin-3-ol (YHR4)
With a four-step conversion using H1 similar to that described for preparation of
YHR17, 8 was converted to YHR4 (18 mg, 29% yield in 4 steps) as a colorless oil.
[α]2D5
1
+15.3 (c = 0.1, MeOH), H NMR (CDCl3, 400 MHz) δ (ppm): 3.52 (t, J = 5.3
Hz, 1H), 3.36 (m, 4H), 3.19 (d, J = 6.6 Hz, 2H), 2.82–2.90 (m, 1H), 2.63 (dd, J = 9.7,
6.2 Hz, 1H), 2.41 (brs, 4H), 1.12–1.79 (m, 25H), 0.90 (q, J = 10.9, 10.1 Hz, 2H). 13C
NMR (CDCl3, 100 MHz) δ (ppm): 76.83, 75.81, 71.13, 65.53, 53.29, 53.00, 38.04,
34.42, 30.18 (2C), 29.74 (2C), 29.55, 29.51, 29.48, 26.90, 26.69, 26.17, 25.90 (2C). IR
(KBr, cm-1): 3427 (br), 3248, 2924, 2850, 2792, 2665, 1463, 1448, 1409, 1375, 1122,
927, 910, 891, 844, 815, 723. HRMS (ESI): calcd for C20H40N2O2H+ [M+H]+ 341.3168,

found 341.3419.
(2R,3S,4S)-4-amino-2-(10-(cyclohexylmethoxy)decyl)pyrrolidin-3-ol (YHR5)
With a four-step conversion using H2 similar to that described for preparation of
YHR17, 8 was converted to YHR5 (11 mg, 17% yield in 4 steps) as a colorless oil.
[α]2D5
+9.4 (c = 0.06, MeOH), 1H NMR (CD3OD, 400 MHz) δ (ppm): 3.63 (t, J = 5.4
Hz, 1H), 3.39 (t, J = 6.5 Hz, 2H), 3.12–3.24 (m, 4H), 2.89 (q, J = 5.9 Hz, 1H), 2.61 (dd,
J = 10.7, 7.2 Hz, 1H), 1.50–1.79 (m, 10H), 1.19–1.45 (m, 21H), 0.94 (q, J = 10.5, 9.0
13
Hz, 2H). C NMR (CD3OD, 100 MHz) δ (ppm): 76.48, 75.83, 70.75, 64.60, 53.31,
50.58, 37.93, 33.51, 29.83 (2C), 29.42, 29.34 (2C), 29.29, 29.27, 29.17, 26.64, 26.37,
26.89, 25.62 (2C). IR (KBr, cm-1): 3338, 3253, 2924, 2850, 2792, 1558, 1541, 1454,
1373, 1124, 964, 916, 887, 815, 721. HRMS (ESI): calcd for C21H42N2O2H+ [M+H]+:
355.3325, found 355.3316.
(2R,3S,4S)-4-amino-2-(11-(cyclohexylmethoxy)undecyl)pyrrolidin-3-ol (YHR6)
With a four-step conversion using H3 similar to that described for preparation of
YHR17, 8 was converted to YHR6 (16 mg, 23% yield in 4 steps) as a colorless oil.
[α]2D5
+15.8 (c = 0.12, MeOH), 1H NMR (CDCl3, 400 MHz) δ (ppm): 3.52 (t, J = 5.3
Hz, 1H), 3.36 (m, 4H), 3.19 (d, J = 6.6 Hz, 2H), 2.86 (d, J = 5.2 Hz, 1H), 2.63 (dd, J =
9.6, 6.1 Hz, 1H), 2.31 (brs, 4H), 1.10–1.81 (m, 29H), 0.90 (q, J = 10.9, 10.1 Hz, 2H).
13C NMR (CDCl3, 100 MHz) δ (ppm): 76.83, 75.80, 71.15, 65.54, 53.26, 52.97, 38.05,
34.40, 30.18 (2C), 29.75 (2C), 29.59 (2C), 29.56, 29.55, 29.50, 26.91, 26.69, 26.18,
25.91 (2C). IR (KBr, cm-1): 3348, 3244, 2922, 2850, 2792, 1558, 1541, 1465, 1448,
1406, 1375, 1124, 1014, 954, 889, 821, 721. HRMS (ESI): calcd for C22H44N2O2H+
[M+H]+ 369.3481, found 369.3477.
(2R,3S,4S)-4-amino-2-(10-isobutoxydecyl)pyrrolidin-3-ol (YHR7)
With a four-step conversion using H4 similar to that described for preparation of
YHR17, 8 was converted to YHR7 (15 mg, 26% yield in 4 steps) as a colorless oil.
[α]2D5
+7.8 (c = 0.06, MeOH), 1H NMR (CD3OD, 600 MHz) δ (ppm): 3.87 (t, J = 5.3
Hz, 1H), 3.52 (q, J = 6.6 Hz, 1H), 3.39 (dt, J = 18.7, 6.7 Hz, 3H), 3.20 (dd, J = 12.6,
6.9 Hz, 3H), 2.93 (dd, J = 11.8, 7.1 Hz, 1H), 1.77–1.89 (m, 1H), 1.69 (dt, J = 15.3, 7.8
Hz, 1H), 1.54 (dt, J = 14.4, 7.2 Hz, 3H), 1.16–1.49 (m, 18H), 0.90 (d, J = 6.7 Hz, 6H).

13C NMR (CD3OD, 150 MHz) δ (ppm): 77.51, 74.02, 70.07, 64.72, 51.99, 31.43, 29.36,
29.32, 29.23, 29.18, 29.15, 29.13, 28.19, 26.32, 25.91,18.37 (2C). IR (KBr, cm-1): 3246,
3070, 2927, 2852, 1635, 1558, 1521, 1419, 1114, 1041, 1008, 968, 945, 873, 831, 819,
721. HRMS (ESI): calcd for C18H38N2O2H+ [M+H]+ 315.3012, found 315.3000.
(2R,3S,4S)-4-amino-2-(9-(benzyloxy)nonyl)pyrrolidin-3-ol (YHR8)
With a four-step conversion using H5 similar to that described for preparation of
YHR17, 8 was converted to YHR8 (23 mg, 37% yield in 4 steps) as a white solid.
[α]2D5
+14.8 (c = 0.11, MeOH), 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.23–7.41 (m,
5H), 4.50 (s, 2H), 3.48 (dt, J = 21.9, 6.1 Hz, 3H), 3.29–3.37 (m, 2H), 2.85 (q, J = 5.8
Hz, 1H), 2.61 (dq, J = 11.7, 6.5, 5.7 Hz, 1H), 2.31 (brs, 4H), 1.06–1.68 (m, 16H). 13C
NMR (CDCl3, 100 MHz) δ (ppm): 138.71, 128.33 (2C), 127.61 (2C), 127.45, 75.84,
72.85, 70.53, 66.51, 53.30, 53.06, 34.47, 29.77, 29.74, 29.53, 29.50, 29.46, 26.90, 26.18.
IR (KBr, cm-1): 3346, 3242, 2924, 2850, 2659, 2536, 1585, 1496, 1454, 1363, 1311,
1114, 1074, 1035, 964, 921, 910, 875, 825, 734, 698, 634, 609. HRMS (ESI): calcd for
C20H34N2O2H+ [M+H]+ 335.2699, found 335.2696.
(2R,3S,4S)-4-amino-2-(10-(1-phenylethoxy)decyl)pyrrolidin-3-ol (YHR9)
With a four-step conversion using H6 similar to that described for preparation of
YHR17, 8 was converted to YHR9 (23 mg, 34% yield in 4 steps) as a colorless oil.
[α]2D5
+16.2 (c = 0.12, MeOH), 1H NMR (CD3OD, 400 MHz) δ (ppm): 7.23–7.34 (m,
5H), 4.40 (q, J = 6.5 Hz, 1H), 3.63 (t, J = 5.3 Hz, 1H), 3.30 (dt, J = 13.4, 7.6 Hz, 3H),
3.18 (ddd, J = 17.6, 11.9, 6.6 Hz, 2H), 2.89 (q, J = 5.8 Hz, 1H), 2.60 (dd, J = 10.6, 7.1
Hz, 1H), 1.08–1.66 (m, 24H). 13C NMR (CD3OD, 100 MHz) δ (ppm): 143.88, 128.07
(2C), 127.10, 125.82 (2C), 77.94, 75.91, 68.37, 64.59, 53.38, 50.72, 33.68, 29.55, 29.45,
29.32, 29.30, 29.29, 29.15, 26.66, 25.90, 23.15. IR (KBr, cm-1): 3344, 3267, 3028, 2926,
2852, 1647, 1558, 1541, 1456, 1406, 1369, 1280, 1207, 1105, 815, 759, 700, 611, 559.
HRMS (ESI): calcd for C22H38N2O2H+ [M+H]+ 363.3012, found 363.2998.
(2R,3S,4S)-4-amino-2-(10-(1-(4-fluorophenyl)ethoxy)decyl)pyrrolidin-3-ol (YHR10)
With a four-step conversion using H7 similar to that described for preparation of
YHR17, 8 was converted to YHR10 (27 mg, 38% yield in 4 steps) as a colorless oil.

[α]2D5
+13.7 (c = 0.12, MeOH), 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.27 (t, J = 6.7
Hz, 2H), 7.02 (t, J = 8.6 Hz, 2H), 4.36 (q, J = 6.3 Hz, 1H), 3.52 (t, J = 4.8 Hz, 1H), 3.35
(dq, J = 16.2, 6.7 Hz, 2H), 3.25 (q, J = 9.0, 7.6 Hz, 2H), 2.80–2.89 (m, 1H), 2.56–2.69
13
(m, 1H), 1.03–1.71 (m, 25H). C NMR (CDCl3, 100 MHz) δ (ppm): 162.03 (d, J =
243.0Hz), 140.03, 127.65 (d, J = 7.0Hz, 2C), 115.14 (d, J = 21.0Hz, 2C), 77.18, 75.81,
68.73, 65.53, 53.29, 53.01, 34.44, 29.91, 29.75, 29.55, 29.54 (2C), 29.43, 26.90, 26.17,
24.20. IR (KBr, cm-1): 3400 (br), 3253, 2926, 2854, 1645, 1606, 1560, 1510, 1456,
1369, 1340, 1224, 1103, 1016, 887, 837, 725, 640, 580. HRMS (ESI): calcd for
C22H37FN2O2H+ [M+H]+ 381.2917, found 381.2761.
(2R,3S,4S)-4-amino-2-(10-((4-fluorobenzyl)oxy)decyl)pyrrolidin-3-ol (YHR11)
With a four-step conversion using H8 similar to that described for preparation of
YHR17, 8 was converted to YHR11 (18 mg, 26% yield in 4 steps) as a colorless oil.
[α]2D5
+14.3 (c = 0.11, MeOH), 1H NMR (CD3OD, 400 MHz) δ (ppm): 7.29–7.38 (m,
2H), 7.05 (t, J = 8.7 Hz, 2H), 4.46 (s, 2H), 3.63 (t, J = 5.3 Hz, 1H), 3.47 (t, J = 6.5 Hz,
2H), 3.18 (ddd, J = 17.5, 12.0, 6.7 Hz, 2H), 2.89 (q, J = 5.8 Hz, 1H), 2.61 (dd, J = 10.7,
7.2 Hz, 1H), 1.20–1.67 (m, 22H). 13C NMR (CD3OD, 100 MHz) δ (ppm): 162.32 (d, J
= 242.0Hz), 134.50, 129.35 (d, J = 8.0Hz, 2C), 114.63 (d, J = 21.0Hz, 2C), 75.85,
71.71, 70.08, 64.60, 53.33, 50.62, 33.58, 29.41, 29.35, 29.31, 29.27, 29.26, 29.14, 26.63,
25.88. IR (KBr, cm-1): 3348, 3242, 2922, 2850, 2791, 2659, 2528, 1514, 1465, 1363,
1298, 1240, 1120, 1016, 966, 921, 879, 821, 767, 721, 634, 567. HRMS (ESI): calcd
for C21H35FN2O2H+ [M+H]+ 367.2761, found 367.2761.
(2R,3S,4S)-4-amino-2-(10-((2,3,5,6-tetrafluoro-4-methylbenzyl)oxy)decyl)pyrrolidin-
3-ol (YHR12)
With a four-step conversion using H9 similar to that described for preparation of
YHR17, 8 was converted to YHR12 (27 mg, 33% yield in 4 steps) as a white solid.
[α]2D5
1
+12.1 (c = 0.13, MeOH), H NMR (CD3OD, 600 MHz) δ (ppm): 4.59 (s, 2H),
3.67 (t, J = 5.4 Hz, 1H), 3.49 (t, J = 6.5 Hz, 2H), 3.26 (q, J = 6.7 Hz, 1H), 3.19 (dd, J =
11.2, 6.7 Hz, 1H), 2.91–2.97 (m, 1H), 2.66 (dd, J = 11.3, 7.5 Hz, 1H), 2.28 (s, 3H), 1.59
(ddt, J = 27.5, 14.0, 7.6 Hz, 3H), 1.22–1.49 (m, 19H). 13C NMR (CDCl3, 100 MHz) δ
(ppm): 146.20, 145.92, 143.86, 143.56, 116.25 (t, J = 19.0Hz, 1C), 113.64 (t, J = 18.0Hz,

1C), 75.81, 70.90, 65.54, 59.74, 53.31, 52.99, 34.43, 29.74, 29.55, 29.52 (3C), 29.37,
26.89, 25.99, 7.61. IR (KBr, cm-1): 3350, 3244, 2920, 2850, 2686, 2538, 1633, 1581,
1487, 1409, 1367, 1284, 1109, 1072, 1031, 956, 923, 875, 837, 752, 719, 667, 597.
HRMS (ESI): calcd for C22H34F4N2O2H+ [M+H]+ 435.2635, found 435.2453.
(2R,3S,4S)-4-amino-2-(10-((4-(trifluoromethyl)benzyl)oxy)decyl)pyrrolidin-3-ol
(YHR13)
With a four-step conversion using H10 similar to that described for preparation of
YHR17, 8 was converted to YHR13 (27 mg, 35% yield in 4 steps) as a white solid.
[α]2D5
+16.1 (c = 0.0.13, MeOH), 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.60 (d, J = 8.0
Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H), 4.55 (s, 2H), 3.50 (dt, J = 19.5, 6.0 Hz, 3H), 3.30–
3.43 (m, 2H), 2.87 (d, J = 5.3 Hz, 1H), 2.64 (dd, J = 9.7, 6.1 Hz, 1H), 2.40 (brs, 4H),
13
1.11–1.68 (m, 18H). C NMR (CDCl3, 100 MHz) δ (ppm): 142.90, 129.60 (d, J =
32.0Hz), 127.46 (2C), 125.26 (q, J = 3.8Hz, 2C), 122.85, 75.76, 72.02, 70.93, 65.52,
53.14, 52.94, 34.33, 29.73 (2C), 29.56, 29.54 (2C), 29.46, 26.89, 26.17. IR (KBr, cm-
1): 3350, 3240, 2926, 2850, 1622, 1558, 1469, 1406, 1340, 1165, 1124, 1066, 1018,
970, 921, 879, 823, 758, 646, 594. HRMS (ESI): calcd for C22H35F3N2O2H+ [M+H]+:
417.2729, found 417.2721.
(2R,3S,4S)-4-amino-2-(10-((2,4-dimethoxybenzyl)oxy)decyl)pyrrolidin-3-ol (YHR14)
With a four-step conversion using H11 similar to that described for preparation of
YHR17, 8 was converted to YHR14 (14 mg, 19% yield in 4 steps) as a white solid.
[α]2D5
+15.7 (c = 0.09, MeOH), 1H NMR (CD3OD, 600 MHz) δ (ppm): 7.09 (d, J = 8.3
Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 6.38 (dd, J = 8.3, 2.3 Hz, 1H), 4.33 (s, 2H), 3.70 (s,
3H), 3.69 (s, 3H), 3.54–3.57 (m, 1H), 3.36 (t, J = 6.6 Hz, 2H), 3.14 (q, J = 6.7 Hz, 1H),
3.08 (dd, J = 11.2, 6.7 Hz, 1H), 2.82 (q, J = 6.1 Hz, 1H), 2.54 (dd, J = 11.2, 7.5 Hz,
1H), 1.44–1.54 (m, 3H), 1.15–1.40 (m, 19H). 13C NMR (CD3OD, 150 MHz) δ (ppm):
161.01, 158.72, 130.32, 118.56, 103.89, 97.80, 75.63, 69.79, 67.08, 64.62, 54.46, 54.37,
53.16, 50.31, 33.30, 29.36, 29.31, 29.27, 29.25, 29.23, 29.11, 26.58, 25.82. IR (KBr,
cm-1): 3433 (br), 3269, 2926, 2850, 1618, 1510, 1259, 1209, 1138, 1093, 970, 920, 869,
829, 790, 719, 634, 565. HRMS (ESI): calcd for C23H40N2O4H+ [M+H]+ 409.3066,
found 409.2900.

4-(((10-((2R,3S,4S)-4-amino-3-hydroxypyrrolidin-2-yl)decyl)oxy)methyl)benzonitrile
(YHR15)
With a four-step conversion using H12 similar to that described for preparation of
YHR17, 8 was converted to YHR15 (18 mg, 26% yield in 4 steps) as a white solid.
[α]2D5
+15.5 (c = 0.11, MeOH), 1H NMR (CD3OD, 600 MHz) δ (ppm): 7.22 (d, J = 8.0
Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 4.45 (s, 2H), 3.64 (dd, J = 6.0, 4.9 Hz, 1H), 3.47 (t,
J = 6.6 Hz, 2H), 3.14–3.24 (m, 2H), 2.90 (dt, J = 7.2, 5.5 Hz, 1H), 2.62 (dd, J = 11.0,
7.4 Hz, 1H), 1.56–1.64 (m, 3H), 1.26–1.51 (m, 15H). 13C NMR (CD3OD, 150 MHz) δ
(ppm): 137.03, 135.34, 128.56 (2C), 127.59 (2C), 75.89, 72.34, 69.87, 64.59, 50.65,
33.61, 29.41, 29.33, 29.31, 29.27, 29.25, 29.13, 26.63, 25.86, 19.83. IR (KBr, cm-1):
3348, 3242, 2920, 2850, 2657, 2520, 1720, 1587, 1516, 1465, 1367, 1114, 1020, 960,
920, 877, 833, 800, 754, 719, 682, 655, 632, 567. HRMS (ESI): calcd for
C22H35N3O2Na+ [M+Na]+ 396.6080, found 396.6080.
(2R,3S,4S)-4-amino-2-(10-hydroxydecyl)pyrrolidin-3-ol (YHR16)
With a four-step conversion using H13 similar to that described for preparation of
YHR17, 8 was converted to YHR16 (13 mg, 28% yield in 4 steps) as a white solid.
[α]2D5
+20.3 (c = 0.12, MeOH), 1H NMR (CDCl3, 400 MHz) δ (ppm): 3.59 (t, J = 6.6
Hz, 3H), 3.20–3.31 (m, 2H), 3.12 (brs, 5H), 2.90 (q, J = 6.6, 5.9 Hz, 1H), 2.62 (dd, J =
10.1, 6.7 Hz, 1H), 1.08–1.65 (m, 18H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 75.74,
65.00, 62.49, 53.18, 51.84, 34.03, 32.52, 29.50, 29.38, 29.31, 29.29, 29.26, 26.66, 25.63.
IR (KBr, cm-1): 3342, 3248, 2922, 2850, 2659, 2528, 1649, 1558, 1467, 1431, 1354,
1321, 1109, 1062, 1020, 966, 920, 877, 831, 786, 719, 634, 570, 513. HRMS (ESI):
calcd for C14H30N2O2H+ [M+H]+ 259.2386, found 259.2380.
4.5 Cell viability
The viability of four cancer cell lines and one normal cardiac fibroblast cell line
was assessed using the MTT assay after the treatment of YHR1-YHR17, and 2-epi-
jaspine B. YHR1-YHR17 and 2-epi-jaspine B were dissolved in pure DMSO to a
concentration of 10 mM and diluted with the culture medium during the experiment.
Five cell lines (A549, LOVO, A375, HepG2, and HCFB) were plated at a density of
6×103 per well in 96-well plates. After culturing for 24 h, YHR1-YHR17 and 2-epi-