Structure-activity relationship studies and bioactivity evaluation of 1,2,3-triazole containing analogues as a selective sphingosine kinase-2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112713

Sphingosine kinase (SphK) is primarily responsible for the production of Sphingosine-1-phosphate (S1P) that plays an important role in many biological and pathobiological processes including cancer, inflammation, neurological and cardiovascular disorders. Most research has focused on developing inhibitors of SphK1 rather than inhibitors of the other isoform SphK2 which has great importance in several pathophysiologic pathways. Exploration of new analogues for improving the potency and selectivity of SphK2 inhibitors is critical. We now have designed, synthesized, and evaluated eighteen new 1,2,3-triazole analogues for their SphK2 inhibitory activity using a ADP-Glo kinase assay, and explored their in vivo anti-tumor bioactivity. Several compounds including 21c, 21e, 21g, 25e-h, 29a-c have high selectivity for SphK2 over SphK1; compound 21g displayed the highest potency with an IC₅₀ value of 0.23 μM. In addition, three compounds 21a, 21b, and 25b have high anti-tumor activity against U-251 MG human glioblastoma cells. Molecular modeling study was performed to elucidate the polar head group and 1,2,3-triazole pharmacophore impact on the SphK2 selectivity.

Full text of DOI:10.1016/j.ejmech.2020.112713

Journal Pre-proof
Structure-activity relationship studies and bioactivity evaluation of 1,2,3-triazole
containing analogues as a selective sphingosine kinase-2 inhibitors
Vijai Kumar Reddy Tangadanchu, Hao Jiang, Yanbo Yu, Thomas J.A. Graham, Hui
Liu, Buck E. Rogers, Robert Gropler, Joel Perlmutter, Zhude Tu
PII:
S0223-5234(20)30685-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112713
EJMECH 112713
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 April 2020
Revised Date: 10 July 2020
Accepted Date: 29 July 2020
Please cite this article as: V.K. Reddy Tangadanchu, H. Jiang, Y. Yu, T.J.A. Graham, H. Liu, B.E.
Rogers, R. Gropler, J. Perlmutter, Z. Tu, Structure-activity relationship studies and bioactivity evaluation
of 1,2,3-triazole containing analogues as a selective sphingosine kinase-2 inhibitors, European Journal
of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112713.
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Graphical Abstract
Structure-activity relationship studies and bioactivity evaluation of 1,2,3-triazole
containing analogues as a selective sphingosine kinase-2 inhibitors
a
a
a
b
c
Vijai Kumar Reddy Tangadanchu , Hao Jiang , Yanbo Yu , Thomas J. A. Graham , Hui Liu ,
d
a
a, e
a,
Buck E. Rogers , Robert Gropler , Joel Perlmutter , Zhude Tu *
a
Department of Radiology, Washington University School of Medicine, St. Louis, Missouri
6
3110, USA.
b
Department of Radiology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104 , USA.
c
Department of Surgery, University of Cincinnati, Cincinnati, Ohio 45267, USA
d
Department of Radiation Oncology, Washington University School of Medicine, St. Louis,
Missouri 63110, USA.
e
Departments of Neurology, Neuroscience, Physical Therapy and Occupational Therapy,
Washington University School of Medicine, St Louis, Missouri 63110, USA.
A novel class of new selective SphK2 inhibitors have been identified. Compound 21g
displayed the highest SphK2 inhibitory activity, and compound 25b exhibited highest anti-tumor
activity against human malignant glioblastoma tumor U-251 MG cell line.
Standard compou nN dT C( 7)
25b

Structure-activity relationship studies and bioactivity evaluation of 1,2,3-
triazole containing analogues as a selective sphingosine kinase-2 inhibitors
a
a
a
b
Vijai Kumar Reddy Tangadanchu , Hao Jiang , Yanbo Yu , Thomas J. A. Graham , Hui
c
d
a
a, e
a,
Liu , Buck E. Rogers , Robert Gropler , Joel Perlmutter , Zhude Tu *
a
Department of Radiology, Washington University School of Medicine, St. Louis, Missouri
3110, USA.
6
b
Department of Radiology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104 , USA.
c
Department of Surgery, University of Cincinnati, Cincinnati, Ohio 45267, USA.
d
Department of Radiation Oncology, Washington University School of Medicine, St. Louis,
Missouri 63110, USA.
e
Departments of Neurology, Neuroscience, Physical Therapy and Occupational Therapy,
Washington University School of Medicine, St Louis, Missouri 63110, USA.
Total Pages: 53
Schemes: 3
Tables: 3
Figures: 7
*
Address correspondence to:
Zhude Tu, Ph.D.
Department of Radiology
Washington University School of Medicine
Campus Box 8225
5
10 S. Kingshighway Blvd.
St. Louis, MO 63110, USA
Tel: 1-314-362-8487
Fax: 1-314-362-8555
E-mail: tuz@mir.wustl.edu

Abstract
Sphingosine kinase (SphK) is primarily responsible for the production of Sphingosine-1-
phosphate (S1P) that plays an important role in many biological and pathobiological processes
including cancer, inflammation, neurological and cardiovascular disorders. Most research has
focused on developing inhibitors of SphK1 rather than inhibitors of the other isoform SphK2
which has great importance in several pathophysiologic pathways. Exploration of new analogues
for improving the potency and selectivity of SphK2 is highly demand. We now have designed,
synthesized, and evaluated eighteen new 1,2,3-triazole analogues for their SphK2 inhibitory
activity using a ADP-Glo kinase assay and their in vivo anti-tumor bioactivity. Several
compounds including 21c, 21e, 21g, 25e-h, 29a-c have high selectivity for SphK2 over SphK1;
compound 21g displayed the highest potency with an IC value of 0.23 µM. In addition, three
5
0
compounds 21a, 21b, and 25b have the highest anti-tumor activity on cell viability against U-
51 MG human glioblastoma cells. Molecular modeling study was performed to elucidate that
2
polar head group and 1,2,3-triazole pharmacophore impact on the SphK2 selectivity.
Keywords: Sphingosine kinase 2 inhibitors, 1,2,3-Triazole hybrids, Selectivity, Molecular
docking, ADP-Glo, U-251 MG Human glioblastoma cell.
_
_____________________
*Corresponding author. Tel.: +1 314 362 8487; fax: +1 314 362 8555.
E-mail: tuz@mir.wustl.edu (Z. Tu).

1
.
Introduction
In the past twenty years, significant strides have been made targeting the sphingolipid signaling as a
therapeutic strategy due to its involvement in a wide range of cellular processes. The natural bioactive
lipid mediator, sphingosine 1-phosphate (S1P) is a ubiquitous cellular signaling molecule that acts as a
critical regulator of many physiological and pathophysiological processes related to, multiple sclerosis
[1], Alzheimer disease [2, 3], cancer [4-7], and viral infections such as Chikungunya virus [8]. In
particular, S1P affects proliferation, angiogenesis, and cell survival [9, 10]. The balance between S1P
and pro-apoptotic ceramide plays an important role in numerous cancers such as non-small cell lung
cancer, gastrointestinal cancer, and glioblastoma [11, 12].
Sphingosine kinase (SphK) is an important metabolic enzyme that catalyzes the phosphorylation of
sphingosine to produce S1P. It plays a pivotal role in the regulation of numerous biological processes
through the balance between S1P and ceramides. SphKs exist in two isoforms, SphK1 and SphK2.
While SphK1 is 270 amino acids shorter than SphK2, they share approximately 50% sequence identity
and the overlapping regions of these two proteins are highly conserved (Supplemental Fig 1). On the
other hand, these two isoforms differ in cellular localization and degree of substrate selectivity [13, 14].
In fact, cellular functions of the two isoforms could vary depending on their subcellular localization.
SphK1 is a cytosolic enzyme that promotes cell survival and proliferation [15, 16], whereas some
SphK2 resides in the nucleus [17, 18], but can relocate to the cytosol on phosphorylation [19, 20].
Depending on the subcellular localization, SphK2 can promote either apoptosis or cell proliferation [8,
2
1]. Due to the critical roles of SphKs in the cell and the importance of the S1P metabolic pathway in
various diseases, investigators have focused on the development of dual and specific SphK inhibitors as
a complementary therapeutic strategy. For example, a specific potent SphK1 inhibitors may provide a
therapeutic for oncology [6], whereas, recent studies have just begun to explore the role of SphK2 [22].
Therefore, identification of highly potent and selective SphK2 inhibitors would be of great value as

pharmacological tools to complement ongoing molecular and genetic studies. Such inhibitors will help
unravel the roles of SphK2 in different pathophysiological conditions and explore potential therapeutic
applications by targeting SphK2.
Fig. 1. Representative SphK1, dual SphK1/2, SphK2 inhibitors, and their inhibitory activities.
There are numeric reports of potent SphK1-selective inhibitors and SphK1/SphK2-dual inhibitors
(
Fig. 1). For example, Compound 1 (PF543, K = 3.6 nM, > 100 fold selective), 2 (Amgen 23), 3
i
(SLP7111228), 4 (CB5468139) are the most potent and selective SphK1 inhibitors, and these ligands
provided as valuable molecular tools to investigate the S1P pathway [23-25]. Pitson and his colleagues
have reported compound 5 (MP-A08) with Ki values of 27±3 µM and 6.9±0.8 µM against human
SphK1 and SphK2, respectively [26]. Compound 6 is another potent dual inhibitor developed by

Schnute and coworkers at Pfizer [27] with an IC50 value of <1.7 nM for both SphK1 and SphK2.
Nevertheless, only a limited SphK2 inhibitors had been reported with moderate or low selectivity and
potency. For example, compound 7 (ABC294640) [28], which is currently in phase II clinical trials for
the treatment of pancreatic cancer and solid tumors (NCT01488513), exhibits moderate inhibitory
activity and selectivity towards SphK2 (IC : SphK2 ≈ 60 µM; SphK1 ≥100 µM). Other SphK2
5
0
selective inhibitors have been reported, including 8 (K145) [29], 9 ((R)-FTY720-OMe) [30], and 10
SLP120701) [25], 11 (SG-12) [31], 12 (trans-12b) [32], compound 13 [33], and compound 14 [34].
(
Therefore, new and adaptable chemical scaffolds for selective SphK2 inhibitors will help identifying the
structural requirements for designing new SphK2 inhibitors and investigating their biological functions.
The lack of a small molecules having highly potent (<100 nM) and selective (>100-fold) SphK2
inhibition prompted our interest to develop improved selective SphK2 inhibitors. Herein, we reported
our efforts to explore a novel scaffold SphK2-selective inhibitors including design, synthesis, biological
evaluation, molecular docking and structure-activity relationship analysis of the most potent SphK2-
1
selective inhibitors described to date. All synthesized target compounds were fully characterized by H-
1
3
NMR, C-NMR, and HRMS analysis. Our data suggested that several lead compounds exhibit relative
high potency and high selectivity for SphK2.
2
.
Results and discussion
2
.1. Design and development of selective SphK2 inhibitors
We designed a series of new SphK2 inhibitors (Fig. 2B) based on the structure activity relationship
(SAR) analysis of reported data [33, 34], as well as the reported lead structures of the SphK2 inhibitors
shown in Fig. 1. Santos et al. recently reported SphK2-selective inhibitors 13, and 14 with K values of
i
either 90 nM, or 89 nM [33, 34]. The inclusion of a methylene unit between the 1,2,4-oxadiazole and
head group pyrrolidinyl rings provided a key factor for the inhibitor’s selectivity.

Fig. 2. Head and terminal group modifications towards the development of novel SphK2 inhibitors.
The authors also investigated the effects of different substituents on the phenyl ring in the terminal
region by interposing the “aminothiazole terminal” linkage to an oxadiazole phenyl ring. Apparently,
positioning of substituents attached to terminal region affected the SphK2 potency and selectivity. Using
these strategies, we have designed and prepared a series of new derivatives by incorporating a 1,2,3-
triazole pharmacophore to replace aminothiazole terminal with diverse aryl structures. Further, we
focused on optimizing the head region of the base structure. Hence, the inclusion/exclusion of methylene
unit and guanidino groups in compounds 13 & 14, prompted us to design derivatives 21a-g, 25a-h, and
2
9a-c (Fig. 3) bearing guanidino, and amidino motifs as the polar head substituents.
Fig. 3. Design of novel 1,2,3-triazole based SphK2 selective inhibitors.
2
.2. Synthesis of SphK2 inhibitors
Guanidine, 1,2,4-oxadiazole, and internal phenyl ring moieties reportedly were key features of the
sphingosine kinase inhibitor scaffold [33, 34]. Therefore, in the present study we focused our attention
on the terminal region by appending 1,2,3-triazole pharmacophore to replace aminothiazole with diverse

aryl structures, to study the structure activity relationship. We made a few modifications based on the
reported structure of the potent compound 13 to develop new SphK2 selective ligands. The synthesis of
various substituted 1,2,3-triazole based SphK2 inhibitors as depicted in Scheme 1.
Scheme 1. Synthesis of target compounds 21a-g. Reagents and conditions: (a) DPPA, toluene, rt, 12 h; (b)
hydroxylamine hydrochloride, NaHCO , CH OH, reflux, 8 h; (c) Boc-L-homoproline, HCTU, DIPEA, DMF, rt to
3
3
1
00 °C, 10 h; (d) Substituted alkynes, 1:1 CH
2 2 2 4 2
Cl :H O, CuSO .5H O, (+)-Sodium L-ascorbate, rt, 12 h; (e) 1:1
TFA: CH Cl , rt, 6 h; (f) N,N-di-Boc-1H-pyrazole-1-carboxamidine, DIPEA, CH CN, 50 °C, 10 h; (g) 4N HCl,
2
2
3
CH OH, rt, 3 h.
3
The synthesis of 1,2,3-triazole containing new SphK2 inhibitors commenced from commercially
available 4-(hydroxymethyl)benzonitrile 15. The alcohol functionality was then transformed into azide
1
6 under the condition of 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) and diphenyl phosphoryl azide
DPPA) in 93% yield. 4-(Azidomethyl)benzonitrile 16 was then treated with hydroxylamine
hydrochloride and sodium bicarbonate as a base in methanol under reflux conditions to yield amidoxime
(
1
7, which is further reacted with homoproline using HCTU and Hunig’s base at ~100 °C to afford the
,2,4-oxadiazole moiety 18 with 56% yield.
1

Our next concern was the divergent synthesis of 1,2,3-triazole based SphK2 inhibitors using 18 as a
building block. A copper catalyzed version of the Huisgen azide-alkyne cycloaddition protocol was
employed to synthesize these triazole compounds. According to Huisgen's protocol [35], the click
reaction between compound 18 with different alkynes, namely phenyl acetylene (19a), 1-ethynyl-4-
(
trifluoromethyl)benzene (19b), 2-ethynylpyridine (19c), hex-1-yne (19d), 1-ethynyl-4-methoxybenzene
19e), ethynylcyclopropane (19f), and 4-ethynyl-1-methyl-1H-pyrazole (19g) in presence of
(
4 2 2
CuSO .5H O and sodium ascorbate as a reducing agent using a mixture of DCM:H O (1:1, v/v)
furnished the corresponding 1,2,3-triazole analogs with moderate to good isolated yields (45-89%) [36].
The Boc group in all the above compounds was removed with trifluoroacetic acid and successively
reacted with N,N-di-Boc-1H-pyrazole-1-carboxamidine in presence of Hunig’s base in acetonitrile at 50
°
C to afford di-Boc-protected guanidines 20a-g in low yields. Finally, the desired target compounds had
been successfully achieved by removal of di-Boc group using 4N HCl solution in dioxane. The desired
guanidine based 1,2,3-triazoles derivatives 21a-g were generated with good yields (65-91%) and further
1
13
characterized by H NMR, C NMR, and HRMS analysis.
We made some modifications based on Scheme 1 to develop additional new ligands for SphK2. We
replaced the homoproline motif on the right side of the base structure with proline, while keeping the
same 1,2,3-triazole pharmacophore on the left side, to study the structure activity relationship. We
synthesized various substituted 1,2,3-triazole based SphK2 inhibitors as outlined in Scheme 2. Moving
forward with our proposed strategy, the synthesis of target compounds 25a-h was initiated with
intermediate 17, which is reacted with Boc-L-proline in presence of HCTU and Hunig’s base at 100 °C
to afford the 1,2,4-oxadiazole moiety 22 with high yield (89%).

Scheme 2. Synthesis of target compounds 25a-h. Reagents and conditions: (a) Boc-L-proline, HCTU, DIPEA,
DMF, rt to 100 °C, 10 h; (b) Substituted alkynes, 1:1 CH Cl :H O, CuSO .5H O, (+)-Sodium L-ascorbate, rt, 12
2
2
2
4
2
h; (c) 1:1 TFA: CH
2 2 3
Cl , rt, 6 h; (d) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIPEA, CH CN, 50 °C, 10 h;
(
e) 4N HCl, CH OH, rt, 3 h.
3
The next concern was the divergent synthesis of 1,2,3-triazole based SphK2 inhibitors using 22 as a
building block. According to Huisgen's protocol, the click reaction between compound 22 with different
alkynes, namely phenyl acetylene (23a), 1-ethynyl-4-(trifluoromethyl)benzene (23b), 2-ethynylpyridine
(23c), 1-ethynyl-4-methoxybenzene (23d), 2-ethynylthiophene (23e), hex-1-yne (23f), 1-ethynyl-4-
fluorobenzene (23g), and ethynylcyclopropane (23h), in presence of CuSO .5H O and sodium ascorbate
4
2
as a reducing agent furnished the corresponding 1,2,3-triazole analogs in good isolated yields (56-85%).
The Boc group in all the above compounds was removed with trifluoroacetic acid and successively
reacted with N,N-di-Boc-1H-pyrazole-1-carboxamidine in the presence of Hunig’s base in acetonitrile at
5
0 °C to afford bis-Boc-protected guanidines in low yields. Finally, the desired target compounds 25a-h
had been successfully achieved by the removal of the di-Boc group with 4N HCl solution in dioxane
afforded the desired guanidine based 1,2,3-triazoles derivatives in good yields (51-89%).

Our next design was to modify the polar head group using amidine motif. Once again, we utilized
the key intermediate 17 to synthesis of amidine based 1,2,3-triazole target compounds 29a-c (Scheme
3
).
Scheme 3. Synthesis of target compounds 29a-c. Reagents and conditions: (a) 1-cyanocyclopropanecarboxylic
acid, PyBop, DIPEA, CH Cl , rt, 4 h; (b) TBAF, THF, rt, 2 h; (c) Substituted alkynes, 1:1 CH Cl :H O,
2
2
2
2
2
CuSO .5H O, (+)-Sodium L-ascorbate, rt, 12 h; (d) NaOMe, CH OH, 50 °C, 24 h; (e) NH Cl, 50 °C, 1 h.
4
2
3
4
Compound 17 was coupled with 1-cyano-1-cyclopropanecarboxylic acid by employing PyBOP to
yield amidoxime compound 26. Cyclization of the coupled amidoxime 26 with tetra-n-butylammonium
fluoride (TBAF) gave oxadiazole 27 in 67% yield. We then synthesized the amidine based 1,2,3-triazole
SphK2 inhibitors employing 27 as a building block. Again, the click reaction was conducted between 27
with different alkynes in presence of CuSO .5H O and sodium ascorbate as a reducing agent afforded
4
2
the corresponding 1,2,3-triazole analogues 28a-c in good isolated yields (62-85%). All the above
synthesized 1,2,3-triazoles were further subjected to base-catalysed Pinner conditions [37] to yield the
corresponding desired target compounds as amidines 29a-c in moderate yields (51-59%).
2
.3. In vitro SphK inhibitory activity evaluation and structure-activity relationship analysis
We sought to discover compounds with high potency and high selectivity for SphK2. Therefore, we
first compared the inhibition activity of our synthesized compounds with the recent reported potent
compound 13 [33] against both SphK1 and SphK2 using ADP-Glo Kinase Assay (Promega, Madison,

WI). Traditional lipid kinase assays rely on quantifying the amount of phosphate transfer to lipids using
lipid extraction and thin-layer chromatography [22, 25, 38]. Although these assays are relative
sensitivity and reliable, it is very challenging to separate the product from substrate. They are also
expensive and labour-intensive, and difficult to use for high-throughput screening. Another method such
as anti- Adenosine Diphosphate (ADP) antibody based assay can be easily used for high-through put
screening, but has low dynamic range and limited sensitivity when the concentration of Adenosine
Triphosphate (ATP) is too low [39].
The bioluminescence-based kinase assay such as ADP-Glo assay measures ADP formed from a
kinase reaction with a luciferase. Upon the completion of a kinase reaction, the remaining ATP is
depleted, the ADP produced in the kinase reaction is then converted to ATP and the detection is based
on a sensitive and robust bioluminescence reaction. It has a broad linear range of ATP concentrations
and high dynamic range. To determine the inhibition activity of our compounds, we used the ADP-Glo
kinase assay which was able to detect down to 20% of 1 µM ATP to ADP conversion (Fig. 4A).
A
B
Y = 47.73*X - 218.1
Fig. 4. Evaluation of ADP-Glo kinase assay performance using recombinant human SphK2. (A) ATP-ADP
conversion curve. ATP to ADP standard curves were generated in the kinase buffer to evaluate the linearity of the
assay and to determine the amount of ADP produced from each tested condition; (B) Human recombinant SphK2
showed a dose-response ATP to ADP conversion activity with an EC of 5.08 ng.
50

It combines the sensitivity and throughput for the detection of ATP conversion, and provides a
reliable method to determine the inhibition activity of chemical molecules toward purified kinase, such
as SphK1 or SphK2. Today, the ADP-Glo kinase assay is widely used for library screening of kinase
inhibitors [40], ATP/ADP metabolism and trafficking [41]. This assay is a convenient method for
medium- and high-throughput screening and has an adequate sensitivity to determine the inhibitory
activity for different kinase such as SphK1 and SphK2. In our studies, as shown in Fig. 4B, our data
indicated that the recombinant human SphK2 has dose-response activity of ATP conversion with an
EC50 of 5.08 ng. After incubation with optimizing concentration (125 µM) of each synthesized
compound, all compounds showed significant reduction of SphK2 activity as expected, with most of
them showed more than a 50% reduction. Meanwhile, all new synthesized compounds only showed
moderate to no effect on SphK1 activity (Fig. 5). The results indicated that all compounds are more
favourable to inhibit SphK2 than SphK1 activity, and thus more selective to SphK2. Among all
compounds, 21c, 21e, 21g, 25e-h, 29a-c displayed the highest specificity to SphK2. In contrast, 21b,
2
1d, 21f, and 25c exhibited the lowest specificity to SphK2.
Fig. 5. The inhibition potency and selectivity of all new synthesized compounds toward SphK1 and SphK2
activity. The inhibitory activity of all compounds on SphK1 and SphK2 were measured using sphingosine kinase
assays and presented as % baseline with no inhibitor added (no treatment control), 125 µM of each compound
were tested in triplicate, error bar indicates S.E. All tested new synthesized compounds showed high potency on

SphK2 inhibition (> 50% inhibition) and moderate to low potency on SphK1 inhibition (< 50% inhibition),
indicated that these new compounds are selective for SphK2.
The IC50 values of new compounds toward SphK2 using ADP-Glo assay were tabulated in Table
1
-3. The results for derivatives 21a-g bearing a 1,2,3-triazole at terminal region as well as a methylene
linker between oxadiazole and a guanidine on the polar head are shown in Table 1. Compound 13
served as a reference standard compound for the inhibitory activity determination in our biological
evaluation in vitro [33].
Table 1. SphK2 inhibitory activity of synthesized target compounds 21a-g.
a
Compounds
1a
R
SphK2 IC50 (µM)
0.396
cLogP
1.97
2
2
1b
--
2.92
2
1c
0.452
--
0.86
1.73
2
1d
2
1e
0.640
--
1.96
0.59
2
1f
2
1g
0.234
0.280
0.17
5.18
Compound 13 (std)

a
cLogP was calculated from Chemdraw Professional 19.0; compound 13 served as a reference standard
compound.
Our in vitro SphK2 inhibition data showed that compounds 21b, 21d, 21f with a para-
trifluoromethyl group of phenyl, butyl, and a cyclopropyl substituted groups on the 1,2,3-triazole
terminal region, respectively, resulted no inhibitory activity, also no selective towards SphK2 (Fig. 5).
In contrast, the compounds bearing a phenyl (21a), 2-pyridyl (21c), and para-methoxy group of phenyl
(21e) substituted groups on the 1,2,3-triazole ring exhibited good to moderate inhibitory activities
towards SphK2 with IC values of 0.396 µM, 0.452 µM, 0.640 µM, respectively, when we compared
5
0
with the standard reference compound (IC50 = 0.28 µM). It is noteworthy that the compound having a 1-
methylpyrazole substituted group on the 1,2,3-triazole terminal (21g), exhibited potent SphK2 inhibitory
activity with an IC value of 0.234 µM which was more potent compared to the reference compound 13
5
0
(
IC = 0.28 µM), and high selectivity for SphK2 over SphK1 (Fig. 5).
50
Our further exploration of new analogues focused on the polar head group without having a
methylene linker between oxadiazole and guanidine motif, and the results are shown in Table 2. From
one side, as shown in Scheme 2, we first retained a guanidine group on the polar head region, and
studied the impact of various substituted groups on the 1,2,3-triazole terminal region 25a-h. All
synthesized analogues 25a-h in this series had moderate to high potent SphK2 inhibitory activity, except
compound 25a. Particularly, compounds having heterocyclic substituents (25c&e) on the 1,2,3-triazole
ring demonstrated moderate inhibition towards SphK2 with IC50 values of 0.900 µM, 0.687 µM,
respectively. A further improvement of SphK2 inhibitory activity was observed with the compounds
bearing either an electron-donating (25d: IC = 0.266 µM) or an electron-withdrawing substituted
5
0
groups (25b: IC = 0.359 µM; 25g: IC = 0.391 µM) on the aromatic ring of the 1,2,3-trizole terminal
5
0
50
region. Interestingly, compounds containing non-aromatic substituents on the 1,2,3-trizole motif, such as
compounds 25f&h proved to be the most potent SphK2 inhibitors in this series with IC values of 0.254
5
0

µM, 0.248 µM, respectively. In addition, these two compounds 25f&h also displayed high selective for
SphK2 over SphK1 (Fig. 5).
Table 2. SphK2 inhibitory activity of guanidine-based target compounds 25a-h.
a
Compound
5a
R
SphK2 IC50 (µM)
--
cLogP
1.87
2
2
5b
0.359
0.909
0.266
2.82
0.76
1.86
2
5c
2
5d
2
5e
0.687
0.254
1.77
1.63
2
5f
2
5g
0.391
2.05
2
5h
0.248
0.280
0.49
5.18
Compound 13 (std)
a
cLogP was calculated from Chemdraw Professional 19.0; compound 13 served as a reference standard
compound.

We next investigated the impact of polar head group having an amidine motif, compounds 29a-c
were synthesized and evaluated for their in vitro SphK2 inhibitory activity, and the results are tabulated
in Table 3. Pleasingly, we found that the introduction of an amidine polar head group containing 1,2,3-
triazoles generally favoured SphK2 inhibition with higher selectivity over SphK1. For example, we
observed better improvement in the inhibitory activities of compounds 29a, and 29b with an IC values
5
0
of 0.261 µM, 0.269 µM respectively. However, compound 29c bearing a cyclopropyl substitution on
,2,3-triazole terminal displayed moderate inhibition (IC50 = 0.717 µM), in comparison to reference
1
standard compound 13.
Table 3. SphK2 inhibitory activity of amidine based target compounds 29a-c.
a
Compound
9a
R
SphK2 IC50 (µM)
0.261
cLogP
1.49
2
2
9b
0.269
-0.31
2
9c
0.717
0.280
0.10
5.18
Compound 13 (std)
a
cLogP was calculated from Chemdraw Professional 19.0; compound 13 served as a reference standard
compound.
Finally, from the in vitro SphK2 inhibitory data shown in Table 1-3, we observed the following
structure-activity relationship information: (a) an introduced methylene linker between oxadiazole and
guanidine polar head group, most of the synthesized target compounds did not increase inhibition

towards SphK2, except compound 21g; (b) excluding a methylene linker between oxadiazole and
guanidine polar head group led to potent inhibition and higher selectivity of most compounds against
SphK2. The inhibitory potency is as follows: non-aromatic > aromatic > heterocyclic substitutions on
the terminal region; (c) further, introducing an amidine polar head group increased the inhibitory activity
of compounds 29a and 29b with an IC50 values of 0.261 µM, 0.269 µM respectively with highly
selective towards SphK2. The structure-activity relationship information of this newly synthesized
scaffolds provides a valuable guide for the future design of new potent and selective SphK2 inhibitors.
2
.4. Modeling docking study
In addition to the SAR information derived from the medicinal chemistry efforts outlined above, we
sought to evaluate potential binding modes for compound 29a within SphK2 utilizing a combination of
homology modeling and flexible receptor docking. Our approach was inspired by recent efforts that
have enabled significant improvements in SphK2 inhibitor potency and selectivity [34]. Given the
relative challenges of accurate homology modeling and ligand binding mode prediction, we sought to
investigate the utility of modern tools for identifying possible binding modes for compound 29a [42,
4
3]. Recently, significant advances have enabled more accurate prediction of protein structure
determination via template-based methods through the use iterative Monte Carlo simulations and model
refinement with molecular dynamics [44-46].

Fig. 6. Top-scoring binding pose of compound 29a within the homology model of SphK2. Key interactions and
their respective distances are highlighted in pink (distance is represented in angstroms).
Through the use of I-TASSAR, we constructed a full-length homology model of SphK2 that was then
subjected to induced fit docking and metadynamics to identify likely binding poses for compound 29a
[47]. We opted to employ an approach that yielded a total of six high-scoring (IFD score) non-
degenerate poses as identified by structural interaction fingerprints and following metadynamics, the
top-scoring pose shown in Fig. 6 (Pose Score) was selected for further analysis. The identified binding
pose is comprised of key hydrogen bonding and electrostatic interactions between the polar amidine
head piece of compound 29a and Asp247, Asp344. Additionally, the amidine moiety of compound 29a
participates in a hydrogen bond with the amide backbone of Leu549 which may be further enabled by
the methylene-cyclopropyl ground found on compound 29a. Contacts within the hydrophobic core and
tail region are in line with previous observations and are comprised of hydrophobic and pi-pi
interactions with Leu549, Leu540, Leu580, Phe339, and Phe584 [34]. Detailed analysis of

metadynamics trajectories for compound 29a will be the subject of future studies. We anticipate that
further refinement of this SphK2 model may enable the use of alchemical methods for free energy
calculations with congeneric ligands [48].
2
.5. In vitro biological evaluation of antitumor activities
Recently, increasing evidence indicates that SphK2 is involved in numerous diseases such as
cancer. Inhibition of SphK2 showed promising anti-tumor effects in multiple cancers such as non-small
cell lung cancer, gastrointestinal cancer, breast cancer, and prostate cancer [11]. It is proposed that SphK
inhibitors can be used for treating malignant tumors such as glioblastoma [49], the most aggressive
primary brain neoplasm with a median survival rate less than 1 year [50]. Targeting on SphK2 may
provide an efficient and effective approach to the treatment of tumor, particularly for glioblastoma [51].

Fig. 7. The antitumor activity of new compounds on U-251 MG glioblastoma tumor cells. (A) The antitumor
activities of all compounds on U-251 MG cell viability were measured and presented as % baseline with no
inhibitor added. Each compound at concentration of 125 µM was tested in triplicate, error bar indicates S.E. All
tested compounds are able to reduce glioblastoma cell viability; three compounds 21a, 21b, and 25b caused the
significant reduction of glioblastoma cell viability, suggested three compounds had the strongest antitumor
activity. (B) Representative images of Hoechst 33258 stained cells after the treatments from selected compounds.
Compound 7 (ABC294640) was reported as a well-known SphK2 selective inhibitor, and it is
currently in phase II clinical trials for the treatment of pancreatic cancer and solid tumors through

various mechanisms [52, 53]. To evaluate the anti-tumor activity of these new SphK2 inhibitors, we
compared the antitumor activities of our new compounds with compound 7 using a human malignant
glioblastoma tumor derived U-251 MG cell line. After 24 hours incubation with 125 µM of each
compound, it was found that compound 7 reduced the cell viability of U-251 MG cells at approximately
5
0% as expected (Fig. 7), surprisingly, compounds 21a-b, and 25b were able to tremendously decrease
the viability of U-251 MG cells at 125 µM in 24 hours. Whereas compounds 21e, 25d, 29a-b showed
comparable reduction of viability of U-251 MG cells with compound 7. This result suggests that our
newly synthesized 1,2,3-triazole SphK2 inhibitors can potentially act as anti-tumor agents. At current
stage, the clear pathway of these new compounds anti-tumor activity resulted solitary from the direct
SphK inhibition of SphK2 or the combinations with facts is remain to be illustrated and further detail
investigations will help the clarification.
3
.
Conclusion
In summary, we successfully designed and synthesized a series of new potent and selective SphK2
inhibitors. All synthesized scaffolds containing guanidine/amidine polar head and 1,2,3-triazole terminal
groups. We further investigated the impact of modifying the polar head group as well as terminal region
on the inhibitory activity and selectivity of these new compounds towards SphK2. All these target
compounds were tested for in vitro SphK2 inhibitory activity using a ADP-Glo kinase assay, and
antitumor activity using human malignant glioblastoma tumor derived U-251 MG cell line. The in vitro
data suggested that compounds 21g, 25d, 25f, 25h, 29a, and 29b were potent towards SphK2 with IC50
values of 0.234, 0.266, 0.254, 0.248, 0.261, and 0.269 µM, respectively. Compounds 21c, 21e, 21g, 25e-
h, 29a-c displayed high selectivity for SphK2 versus SphK1. Three compounds 21a-b, and 25b also
exhibited higher antitumor activity for human malignant glioblastoma tumor U-251 MG cell line when
compared with ABC294640. Molecular docking study revealed that polar head group plays an important
role in the participation of hydrogen bonding, electrostatic interactions with Asp247, Asp344, and

Leu549. In addition, 1,2,3-triazole group fits securely into the hydrophobic “tail” region of the SphK2
binding site, thus might be leads to increased potency toward SphK2 with higher selectivity. Overall, our
investigation provides a valuable information for further design and development of high potent and
selective SphK2 inhibitors which may facilitate the development of therapeutics for treating diseases
targeting on SphK2 and related sphingosine-mediated signaling.
4
.
Experimental
4
.1. General
All reagents, and starting materials used in this study were obtained from commercial sources
without any further purification. All dry reactions were conducted under a nitrogen atmosphere in oven
dried glass apparatus using dry solvents. Yields refer to chromatographically, unless otherwise stated.
Reactions were monitored by thin-layer chromatography (TLC) carried out on precoated glass plates of
silica gel (0.25 mm) 60 F254 from EMD Chemicals Inc. Visualization was accomplished with ultraviolet
light (UV 254 nm), or by shaking the plate in a sealed jar containing silica gel and Iodine. Flash column
chromatography was performed using Silia Flash® P60 40-63µm (230-400 mesh) from Silicycle.
1
13
Melting points were determined on a MEL-TEMP 3.0 apparatus. H NMR and C NMR spectra were
1
13
recorded on Varian 400 MHz (operating at 400 MHz for H and 100 MHz for C NMR) spectrometer.
Rotamers are denoted by an asterisk (*). Chemical shifts are reported in parts per million (ppm) and
coupling constants
J are given in Hz (Hertz). Chemical shifts are reported relative to TMS (δ = 0.0) as
an internal standard. (Abbreviations used in spectra: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad, dd = double of doublets, dt = doublet of triplets, td = triplet of doublets, qd =
+
quartet of doublets). High resolution mass spectra (HRMS) [ESI] were recorded on a Bruker MaXis 4G
Q-TOF mass spectrometer with electrospray ionization source.

4
4
.2. Chemistry
.2.1.General procedure A: Coupling of amide-oxime derivatives with (S)-2-(1-(tert-
butoxycarbonyl)pyrrolidin-2-yl)acetic acid/N-(tert-butoxycarbonyl)-L-proline
To a solution of (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid/N-(tert-butoxycarbonyl)-
L-proline (1.1 equiv) in DMF (10 mL) was added DIPEA (1.8 equiv) and followed by addition of HCTU
(1.5 equiv). The reaction mixture was stirred for 0.5 h followed by adding amidoxime (1.0 equiv). The
reaction mixture was stirred for 1 h at room temperature, then stirred at 100 °C oil-bath for 8-10 h. The
reaction progress was monitored by TLC. The solution was partitioned between EtOAc and LiBr
aqueous solution. The aqueous solution was washed with EtOAc (3 × 10 mL), and the combined organic
layers were washed with brine, dried over Na SO , filtered, and concentrated via vacuum. The resulting
2
4
residue was purified by silica gel column chromatography.
4
.2.2.General procedure B: Synthesis of 1,2,3-triazole derivatives
To a vigorously stirred solution of the azide (1 equiv) in DCM (5 mL) was added various terminal
alkynes (1.1 equiv). The reaction was initiated by the addition of a solution of CuSO .5H O (0.01 equiv)
4
2
and sodium ascorbate (0.1 equiv) in distilled water (5 mL). The coloured suspension that formed was
stirred at room temperature for 10-12 h. The reaction progress was monitored by TLC. After completion
of the reaction, the resulting mixture was diluted with CH
combined organic extracts were dried over anhydrous Na SO
pressure to afford crude product, which was purified by silica gel column chromatography to obtain pure
,2,3-triazoles.
2
2
Cl and then washed with brine. The
2
4
and then concentrated under reduced
1

4
.2.3.General procedure C: Deprotection of t-Boc protecting groups with TFA and guanylation of
amines
To a solution of azole based
2 2
t-Boc-protected intermediates (1.0 equiv) in CH Cl (5 mL) was added
a TFA (6.0 equiv) in CH Cl (5 mL). The reaction mixture was then stirred at rt for 4-6 h, and monitored
2
2
via TLC. At this time, TLC showed complete conversion of the starting material. The organic solvent
was removed under reduced pressure. The residue was then dissolved in CH CN (10 mL).
3
Diisopropylethylamine (10 equiv) and (Z)-tert-butyl (((tert-butoxycarbonyl)imino)(1H-pyrazol-1-
yl)methyl)carbamate (1.1 equiv) were added to the above solution, and the resulting reaction mixture
was stirred at 50 °C for 8-10 h, and monitored via TLC. Once the starting material was consumed, the
solution was cooled to room temperature, and the solvent was removed under reduced pressure to afford
the crude residue. The resulting residue was purified by silica gel column chromatography with 40-50%
EtOAc in hexane to afford the desired 1,2,3-triazole based guanylated derivatives.
4
.2.4.General procedure D: Deprotection of t-Boc protecting groups with HCl
To a solution of
N-Boc-protected compounds (1.0 equiv) in methanol (1 mL) was then added 4N
hydrochloric acid (HCl) solution in dioxane (1 mL). The resulting reaction mixture was stirred for 2-3 h
and monitored via TLC. Once the starting material was consumed, and the solvent was removed under
reduced pressure to get the residue. The resulting residue was triturated with diethyl ether to yield the
corresponding free amine hydrochloride salt.
4
.2.5.General procedure E: Conversion of nitriles to amidines
To a solution of a nitrile (1.0 eq.) in MeOH (0.10 M) was added a 0.5 M solution of sodium
methoxide in MeOH (0.50 eq.) at rt and then heated to 50 °C for 24 h. The intermediate imidate was
detectable by TLC; however, being in equilibrium with the nitrile, full conversion does not occur.
Ammonium chloride (4.0 eq.) was then added in one portion at that temperature and allowed to react

until the imidate was completely consumed by TLC analysis. The reaction was then cooled to rt and
evacuated to dryness to yield a crude solid. The solid was dissolved in minimum amount of water in
order to remove excess ammonium chloride, and the solid was again evacuated to dryness. The crude
solid material was then recrystallized in Et O and DCM to yield the pure amidine hydrochloride salt.
2
The yields varied greatly depending upon substrate, because amidine formation is dependent upon the
equilibrium ratio between nitrile and imidate established under the sodium methoxide conditions.
4
.2.6.4-(Azidomethyl)benzonitrile (16)
To a round-bottom flask equipped with a stir bar was added 4-(hydroxymethyl)benzonitrile (5.0 g,
7.55 mmol), diphenyl phosphoryl azide (9.68 mL, 45.06 mmol), and toluene (25 mL). The mixture was
3
cooled to 0 °C before adding 1,8- diazabicyclo [5.4.0]undec-7-ene (6.72 mL, 45.06 mmol) dropwise.
The reaction was warmed to room temperature slowly and continued to stir for overnight. The reaction
progress was monitored by TLC. After completion of the reaction, the resulting mixture was diluted
with water (30 mL) and extracted with ethyl acetate (30 mL). The ethyl acetate layer was washed with
1
2 4
M HCl, saturated brine and dried over anhydrous Na SO . The organic solvent was removed under
reduced pressure. The crude residue was purified on a silica gel column chromatography, eluted with
1
1
0% EtOAc in hexane to afford the desired compound 16 (5.52 g, 93% yield) as a colorless solid. H
NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 4.42 (s, 2H).
4
.2.7.4-(Azidomethyl)-N'-hydroxybenzimidamide (17)
To a stirred suspension of NH
2 3
OH·HCl (4.39 g, 63.22 mmol) and NaHCO (10.62 g, 126.44 mmol)
in CH OH (50 mL), a solution of 16 (5.0 g, 31.61 mmol) in CH OH (10 mL) was gradually added. The
3
3
reaction was refluxed and stirred in a pre-heated 75 °C oil-bath for 8 h. The reaction progress was
monitored by TLC. After completion of the reaction, it was cooled to room temperature, and the
precipitate was filtered off and washed with methanol. The filtrate was concentrated in vacuo without

1
further purification to afford 17 (5.50 g, 91% yield) as a white solid. H NMR (400 MHz, DMSO-d6)
δ
9
.68 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 5.83 (s, 2H), 4.46 (s, 2H).
4
.2.8.(S)-tert-Butyl-2-((3-(4-(azidomethyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-
carboxylate (18)
1
Synthesized by general procedure A: Yield: 56%; Pale yellow color solid; H NMR (400 MHz,
CDCl
3
)
δ
8.08 (d,
J
= 8.2 Hz, 2H), 7.41 (d,
2.98 (m, 1H), 2.12
J
= 8.0 Hz, 2H), 4.40 (s, 2H), 4.28 (d,
J = 24.9 Hz, 1H), 3.45
‒
3.28 (m, 3H), 3.15
‒
‒
2.03 (m, 1H), 1.90 1.78 (m, 3H), 1.45 (s, 9H).
‒
4
.2.9.(S)-tert-Butyl-2-((3-(4-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1,2,4-oxadiazol-5-
yl)methyl)pyrrolidine-1-carboxylate (19a)
1
Synthesized by general procedure B: Yield: 81%; White solid; H NMR (400 MHz, CDCl
3
)
δ
8.08
(
d,
J
= 8.1 Hz, 2H), 7.79 (d,
.62 (s, 2H), 4.27 (d, = 31.0 Hz, 1H), 3.43-3.27 (m, 3H), 3.15
.91 1.77 (m, 3H), 1.44 (s, 9H).
J
= 7.4 Hz, 2H), 7.69 (s, 1H), 7.42
‒
7.36 (m, 4H), 7.30 (t, = 7.3 Hz, 1H),
J
5
1
J
‒
2.97 (m, 1H), 2.10 ‒ 2.03 (m, 1H),
‒
4
.2.10. (S)-tert-Butyl-2-((3-(4-((4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-
,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (19b)
1
1
Synthesized by general procedure B: Yield: 45%; White solid; H NMR (400 MHz, CDCl
= 8.2 Hz, 2H), 7.90 (d, = 8.1 Hz, 2H), 7.76 (s, 1H), 7.64 (d, = 8.2 Hz, 2H), 7.40 (d, = 8.0 Hz,
= 31.4 Hz, 1H), 3.45 3.27 (m, 3H), 3.12 2.98 (m, 1H), 2.09 1.99 (m,
1.77 (m, 3H), 1.44 (s, 9H).
3
) δ 8.09
(
d,
J
J
J
J
2
1
H), 5.64 (s, 2H), 4.27 (d,
J
‒
‒
‒
H), 1.89
‒
4
.2.11. (S)-tert-Butyl-2-((3-(4-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1,2,4-oxadiazol-5-
yl)methyl)pyrrolidine-1-carboxylate (19c)

1
Synthesized by general procedure B: Yield: 78%; White solid; H NMR (400 MHz, CDCl
= 4.8 Hz, 1H), 8.15 (d, = 7.9 Hz, 1H), 8.10 8.03 (m, 3H), 7.74 (td, = 7.6, 1.2 Hz, 1H), 7.40 (d,
= 8.0 Hz, 2H), 7.19 (dd, = 6.6, 5.1 Hz, 1H), 5.62 (s, 2H), 4.26 (d, = 29.4 Hz, 1H), 3.42 3.37 (m,
H), 3.13 2.96 (m, 1H), 2.09 2.03 (m, 1H), 1.90 1.75 (m, 3H), 1.43 (s, 9H).
3
) δ 8.51
(
d,
J
J
‒
J
J
J
J
‒
3
‒
‒
‒
4
.2.12. (S)-tert-Butyl-2-((3-(4-((4-butyl-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1,2,4-oxadiazol-5-
yl)methyl)pyrrolidine-1-carboxylate (19d)
1
Synthesized by general procedure B: Yield: 85%; White solid; H NMR (400 MHz, CDCl )
δ
8.05
= 33.0 Hz, 1H), 3.45
2.05 (m, 1H), 1.92 1.76 (m, 3H),
= 7.3 Hz, 3H).
3
(
d,
J
= 8.1 Hz, 2H), 7.32 (d,
.26 (m, 3H), 3.12 3.00 (m, 1H), 2.68 (t,
.65 1.57 (m, 2H), 1.44 (s, 9H), 1.37 1.31 (q, 2H), 0.89 (t,
J
= 8.0 Hz, 2H), 7.19 (s, 1H), 5.53 (s, 2H), 4.26 (d,
J
‒
3
1
‒
J
= 7.7 Hz, 2H), 2.10
‒
‒
‒
‒
J
4
.2.13. (S)-tert-Butyl-2-((3-(4-((4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1,2,4-
oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (19e)
1
Synthesized by general procedure B: Yield: 57%; White solid; H NMR (400 MHz, CDCl )
δ 8.11
3
(
d,
J
= 8.2 Hz, 2H), 7.75 (d,
H), 5.64 (s, 2H), 4.31 (d,
2.04 (m, 1H), 1.93 1.78 (m, 3H), 1.48 (s, 9H).
J
= 8.8 Hz, 2H), 7.64 (s, 1H), 7.42 (d,
J
= 8.0 Hz, 2H), 6.96 (d,
J = 8.8 Hz,
2
‒
J
= 36.5 Hz, 1H), 3.85 (s, 3H), 3.46 3.32 (m, 3H), 3.16 ‒
‒
3.02 (m, 1H), 2.13
‒
4
.2.14. (S)-tert-Butyl-2-((3-(4-((4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1,2,4-oxadiazol-5-
yl)methyl)pyrrolidine-1-carboxylate (19f)
1
Synthesized by general procedure B: Yield: 89%; White solid; H NMR (400 MHz, CDCl )
δ
8.11
3
(
d,
J
= 8.1 Hz, 2H), 7.38 (d,
.34 (m, 3H), 3.23 3.04 (m, 1H), 2.14
0.85 (m, 2H).
J
= 8.0 Hz, 2H), 7.22 (s, 1H), 5.57 (s, 2H), 4.33 (d,
J
= 27.4 Hz, 1H), 3.53
‒
3
‒
‒
2.11 (m, 1H), 2.00 1.88 (m, 4H), 1.50 (s, 9H), 1.02
‒
‒
0.94
(m, 2H), 0.89
‒

4
.2.15. (S)-tert-Butyl-2-((3-(4-((4-(1-methyl-1H-pyrazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-
,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (19g)
1
1
Synthesized by general procedure B: Yield: 51%; White solid; H NMR (400 MHz, CDCl
= 7.9 Hz, 2H), 7.80 (s, 1H), 7.72 (s, 1H), 7.51 (s, 1H), 7.41 (d, = 7.7 Hz, 2H), 5.62 (s, 2H), 4.30
= 27.6 Hz, 1H), 3.95 (s, 3H), 3.47 3.31 (m, 3H), 3.14 3.00 (m, 1H), 2.11
3
) δ 8.11
(
d,
d,
J
J
J
(
‒
‒
‒
2.02 (m, 1H), 1.94
‒
1
.80 (br m, 3H), 1.47 (s, 9H).
4
.2.16. (S)-tert-Butyl-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-phenyl-1H-1,2,3-triazol-1-
yl)methyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (20a)
1
Synthesized by general procedure C: Yield: 51%; White solid; H NMR (400 MHz, CDCl
3
)
δ
10.27
(
s, 1H), 8.10 (d,
J
= 8.0 Hz, 2H), 7.79 (d,
= 7.3 Hz, 1H), 5.62 (s, 2H), 4.77
3.13 (m, 1H), 2.27 2.20 (m, 1H), 1.91
J
= 7.6 Hz, 2H), 7.67 (s, 1H), 7.39 (dd,
4.73 (m, 1H), 3.66 3.59 (m, 2H), 3.50
1.87 (m, 1H), 1.82 1.73 (m, 2H), 1.44 (s, 18H).
J
= 7.4, 5.1 Hz, 4H),
7
3
.30 (t,
.19
J
‒
‒
‒
3.41 (m, 1H),
‒
‒
‒
‒
4
.2.17. (S)-tert-Butyl-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-
triazol-1-yl)methyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (20b)
1
Synthesized by general procedure C: Yield: 60%; White solid; H NMR (400 MHz, CDCl )
δ
10.38
= 8.0 Hz, 2H), 7.48 (d,
= 17.2, 7.9 Hz, 2H), 3.59 3.49 (m, 1H),
1.94 (m, 1H), 1.90 1.81 (m, 2H), 1.52 (s,
3
(
s, 1H), 8.19 (d,
J
= 7.8 Hz, 2H), 7.98 (d,
= 7.9 Hz, 2H), 5.72 (s, 2H), 4.85 4.82 (m, 1H), 3.71 (dd,
2.27 (m, 1H), 1.98
J = 7.9 Hz, 2H), 7.83 (s, 1H), 7.72 (d, J
J
3
1
‒
J
‒
.24 (dd,
8H).
J
= 14.4, 8.5 Hz, 1H), 2.34
‒
‒
‒