Identification of novel and selective non-peptide inhibitors targeting the polo-box domain of polo-like kinase 1

Article abstract of DOI:10.1016/j.bioorg.2018.08.030

A series of non-peptide inhibitors targeting the polo-box domain (PBD) of polo-like kinase 1 (Plk1) was designed based on the potent and selective minimal tripeptide Plk1 PBD inhibitor. Seven compounds were designed, synthesized and evaluated for fluorescence polarization (FP) assay. The most promising compound 10 bound to Plk1 PBD with IC₅₀ of 3.37 μM and had no binding to Plk2 PBD or Plk3 PBD at 100 μM. Molecular docking study was performed and possible binding mode was proposed. MM/GBSA binding free energy calculation were in agreement with the observed experimental results. These novel non-peptide selective Plk1 PBD inhibitors provided new lead compounds for further optimization.

Full text of DOI:10.1016/j.bioorg.2018.08.030

Accepted Manuscript
Identification of Novel and Selective Non-peptide Inhibitors Targeting the Polo-
Box Domain of Polo-Like Kinase 1
Yanhong Chen, Zhiyan Li, Yu Liu, Tongyuan Lin, Huiyong Sun, Dasong Yang,
Cheng Jiang
PII:
S0045-2068(18)30765-X
https://doi.org/10.1016/j.bioorg.2018.08.030
YBIOO 2487
DOI:
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 July 2018
15 August 2018
21 August 2018
Please cite this article as: Y. Chen, Z. Li, Y. Liu, T. Lin, H. Sun, D. Yang, C. Jiang, Identification of Novel and
Selective Non-peptide Inhibitors Targeting the Polo-Box Domain of Polo-Like Kinase 1, Bioorganic Chemistry
(2018), doi: https://doi.org/10.1016/j.bioorg.2018.08.030
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Identification of Novel and Selective Non-peptide Inhibitors Targeting the
Polo-Box Domain of Polo-Like Kinase 1
c
Yanhong Chen a,b,c, Zhiyan Li a,b,c, Yu Liu a,b,c, Tongyuan Lin a,b,d, Huiyong Sun ,
Dasong Yang a,b,d,*, Cheng Jiang a,b,c,*
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, Nanjing 210009, China
b Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical
University, Nanjing 210009, China
c
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing
210009, China
d
Key Laboratory of Drug Quality Control and Pharmacovigilance, China
Pharmaceutical University, Nanjing 210009, China
* Corresponding authors at: Jiangsu Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China.
E-mail addresses: yangds@cpu.edu.cn (D. Yang), jc@cpu.edu.cn (C. Jiang).

Abstract:
A series of non-peptide inhibitors targeting the polo-box domain (PBD) of polo-like
kinase 1 (Plk1) was designed based on the potent and selective minimal tripeptide
Plk1 PBD inhibitor. Seven compounds were designed, synthesized and evaluated for
fluorescence polarization (FP) assay. The most promising compound 10 bound to Plk1
PBD with IC50 of 3.37 μM and had no binding to Plk2 PBD or Plk3 PBD at 100 μM.
Molecular docking study was performed and possible binding mode was proposed.
MM/GBSA binding free energy calculation were in agreement with the observed
experimental results. These novel non-peptide selective Plk1 PBD inhibitors provided
new lead compounds for further optimization.
Key words: Polo like kinase 1; Polo-box domain; Inhibitors; Non-peptide; Molecular
docking

1. Introduction
Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase which acts as a key
regulator in multiple stages of mitotic progression [1]. Plk1 modulates the transition
through the G2/M checkpoint by influencing the activation of the CDC25C
phosphatase and cyclin B1 [2]. The overexpression of Plk1 is required for the viability
of broad spectrum of cancer cells and interference with Plk1 function induces cell
apoptosis in most cancer cells [3]. Therefore Plk1 has been considered as an attractive
anti-cancer drug target for anticancer drug development.
Five Plks in mammalian cells have been identified (Plk1-5) so far. Among them,
Plk1-4 consists of an N-terminal catalytic domain and a C-terminal domain having 1
or 2 highly conserved sequences, termed polo-box domains (PBDs) [4]. A large body
of evidence suggests that PBD directs the N-terminal catalytic domain for specific
subcellular localization through interacting with phosphoserine/phosphothreonine
(pS/pT)-containing motifs. Moreover, the subcellular targeting binding site in PBD
forms a compact and druggable interface [5].
Plk1, Plk2 and Plk3 are three closely related members while Plk4 is a distantly related
kinase with apparently different expression patterns and physiological functions [3,4].
In contrast to the role of Plk1 in cell proliferation and tumorigenesis, the two most
closely related kinases, Plk2 and Plk3, seem to have a role in checkpoint-mediated
cell-cycle arrest to ensure genetic stability and prevent oncogenic transformation [3-5].
Thus, the development of Plk1-specific inhibitors could be important for anti-Plk1
cancer therapy.

For a long period, several potent orthosteric Plk1 inhibitors targeting the ATP-binding
catalytic domain have been reported [3]. However, the ATP-binding sites of the
catalytic domains of protein kinases are closely related, these efforts suffered from a
lack of selectivity. Rigosertib (ON01910) was reported as a Plk1 allosteric inhibitor,
which showed potent Plk1 inhibitory activity and antitumor activity [6-8]. However, it
has an inhibitory effect on multiple target (Plk3, Chk1, Chk2, BCR-ABL, Fyn, Src
and Ras) [9], among which Plk3 was considered as a negative regulator responsible
for the inhibition of cell cycle progression and tumor cell development at multiple
stages of mitosis. Based on the structure of PBIP1 which was isolated as a
PBD-interacting protein crucial for centromeric localization of Plk1, a minimal
peptide (PLHSpT, 1) was identified as an inhibitor with high affinity toward the Plk1
PBD and high selectivity against Plk2 PBD and Plk3 PBD [10].
After the identification of minimal peptide (1) with high affinity toward the Plk1 PBD,
several phosphate peptides and peptidomimetics were reported (Fig. 1, 2-4) [11-19].
Then a series of (2S,3R)-2-amino-3-methyl-4-phosphonobutanoic acid (Pmab)
incorporated peptidomimetic was reported (Fig. 1, 5) to produce a phosphatase-stable
peptidomimetic with complete retention of inhibitory potency [20]. To date, most of
the modification made to peptides and peptidomimetics acting as Plk1 PBD inhibitors
focusing on the 1-3 N-terminal residues of PLHSpT. Very few modification has been
made to the C-terminal SpT part which was considered essential for high-affinity
binding [10]. In this study, we make the modification on the C-terminal SpT residue
based on the highly potent Plk1 tripeptide (Fig. 1, 4) reported [17] and provide new

non-peptide skeleton for further modification toward the discovery and development
of highly potent and selective Plk1 PBD inhibitors.
Fig. 1. Examples of the reported phosphate peptides and peptidomimetics as Plk1 PBD inhibitors.
2. Results and discussion
2.1. Design, synthesis and evaluation
According to the binding mode of 4 with Plk1 PBD (PDB ID: 4WHH), the N-terminal
fragment of peptidomimetic 4 and C6H5(CH2)8-group on the imidazole ring were
directed toward the Tyr-rich hydrophobic channel, whereas the negatively charged
pThr phosphoryl group mediated strong electrostatic interactions with the H538 and
K540 residues [17]. The residues of Ser and Thr in 4 acted as a linker and limited the
direction of the phosphate acid substitution. In our research, the imidazole ring of the
His in 4 was substituted by a triazole ring for easier synthesis (blue in Fig.2). The

linker formed by Ser and Thr was substituted by a rigid linker (red in Fig.2) which
may also limit the direction of the terminal phosphate acid. It was reported that Pmab
incorporated peptidomimetic could produce a phosphatase-stable analogue of
phosphothreonine (pThr) [20]. Thus 3-phosphonopropanoic acid was attached to the
aromatic ring as compound 5. Then compound 6, 7 and 8 were designed as shown in
Fig.2.
Fig. 2. Design strategy of compound 6, 7 and 8 with new skeleton based on 4.
The key intermediate 22 was synthesized using the method as shown in Scheme 1 and
Scheme 2. Compound 6 was synthesized according to the route shown in Scheme 3
and Scheme 4. The intermediate of 7 and 8 was prepared according to the method as
shown in Scheme 5. And the target compound 7 and 8 were readily obtained using the
route in Scheme 8.

Scheme 1. Reagents and conditions: (i) MsCl, Et3N, DCM, rt, 2 h; (ii) NaN3, DMF, 80℃, 5 h; (iii)
K2CO3, BnBr, DMF, rt, 1 h; (iv) Cp*RuCl(PPh3)2, DCE, reflux, 12 h.
Scheme 2. Reagents and conditions: (i) EDCI, HOBt, Et3N, DCM, rt, 5 h; (ii) 3N NaOH,
1,4-dioxane, rt, 2 h; (iii) 17, EDCI, HOBt, Et3N, DCM, rt, 5 h; (iv) H2, 10% Pd/C, MeOH, rt, 40
min.
Scheme 3. Reagents and conditions: (i) Ac2O, Et3N, toluene, 80 ℃, 1 h; (ii) HNO3, Ac2O, 0℃, 1

h; (iii) 10N NaOH, H2O, MeOH, 15 min; (iv) HBr, reflux, 10 h; (v) Triethyl phosphite, reflux, 5 h;
(vi) H2, 10 % Pd/C, MeOH, r.t., 4 h.
Scheme 4. Reagents and conditions: (i) HATU, HOBt, DIPEA, DCM, rt, 3 h; (ii) Ac2O, 65℃, 6 h;
(iii) TMSBr, DCM, rt, 10 h.
Scheme 5. Reagents and conditions: (i) Boc2O, NaOH, NaHCO3, rt, 5 h; (ii) MsCl, Et3N, rt, 2 h;
(iii) NaBr, TBAB, DMF, 36 h; (iv) Dibenzyl phosphite, TBAI, Cs2CO3, DMF, rt, 10 h; (v) TFA,
DCM, rt, 0.5 h.
The binding affinity of 6, 7 and 8 to Plk1 PBD was evaluated using our optimized
fluorescein polarization (FP) binding assay [21]. As shown in Table 1, none of the
three compound (6, 7 and 8) showed binding affinity to Plk1 PBD. The binding of the
pT residue is essential for high-affinity binding, a slight change may cause complete
loss of binding. Further modification was focused on how to adjust the direction of
the 3-phosphonopropanoic acid side chain. Triazole group was introduced to mimic
the aromatic ring (red in Fig.2) linked with 3-phosphonopropanoic acid.

Appropriate azide was reacted with alkyne in the presence of copper sulfate and
sodium ascorbate (VcNa) to give the protected triazolyl contained intermediate
(Scheme 6). The alternative 1,5-regioisomer was achieved using the ruthenium
catalyst Cp*RuCl(PPh3)2 (Scheme 7) [22,23]. Then compound 9 and 10 were
synthesized using the similar method as 7 and 8 as shown in Scheme 8.
Scheme 6. Reagents and conditions: (i) NaH, BnBr, 0℃, 2 h; (ii) MsCl, Et3N, rt, 2 h; (iii) TMSN3,
K2CO3, DMF, 100 ℃ , 5 h; (iv) N-Boc-propargylamine, CuSO4 , sodium ascorbate,
t-BuOH:H2O= 1:1, 3 h; (v) H2, Pd/C, MeOH, rt, 5 h; (vi) MsCl, Et3N, rt, 2 h; (vii) NaBr, TBAB,
DMF, 36 h; (viii) Dibenzyl phosphite, TBAI, Cs2CO3, DMF, rt, 10 h; (ix) TFA, DCM, rt, 0.5 hr.
Scheme 7. Reagents and conditions: (i) N-Boc-propargylamine, Cp*RuCl(PPh3) 2, DCE, reflux, 5
h; (ii) H2, Pd/C, MeOH, rt, 5 h; (iii) MsCl, Et3N, rt, 2 h; (iv) NaBr, TBAB, DMF, 36 h; (v)

Dibenzyl phosphite, TBAI, Cs2CO3, DMF, rt, 10 h; (vi) TFA, DCM, rt, 0.5 h.
Scheme 8. Reagents and conditions: (i) HATU, HOBt, DIPEA, DCM, rt, 3 h; (ii) H2, 10% Pd/C,
MeOH, rt, 40 min; (iii) CF3COOH, rt, 1 h.
The binding affinity of 9 and 10 to Plk1 PBD was evaluated using FP assay. As
shown in Table 1, the 1,5-regioisomer 9 showed no binding affinity to Plk1 PBD,
while the 1,4-regioisomer 10 showed moderate Plk1 PBD binding affinity (IC50 =
3.37 μM). Thus the 1,4-regioisomer 10 was selected for further exploration.
Compounds with different length of the linker between the triazolyl and the
phosphonic acid was made to find the optimal length. Compound 11 and 12 was

synthesized using the same method as 10. The binding affinity of 11 and 12 was
evaluated using FP assay. As shown in Table 1, 11 showed similar Plk1 PBD binding
affinity (IC50 = 5.45 μM) as 10, while the binding affinity of 12 was slightly
decreased (IC50 = 11.12 μM), suggesting that the 1,4-regioisomer triazolyl attached
with ethyl or propyl could be an optimal length of linker for the phosphonic acid. The
binding affinity of 10, 11 and 12 with Plk2 PBD and Plk3 PBD was also evaluated
using FP assay. As shown in Table 1, none of the tested compounds showed binding
affinity to Plk2 PBD and Plk3 PBD. The new identified non-peptide inhibitors
showed excellent selectivity to Plk1 PBD against Plk2 PBD and Plk3 PBD.
Table 1. Structures of designed compounds and their binding affinity to Plk1 PBD,
Plk2 PBD and Plk3 PBD
Plk1 PBD
Plk2 PBD
Plk3 PBD
Compound
6
R
IC50 (μM)a
IC50 (μM)a
IC50 (μM)a
>100
>100
ND
ND
ND
ND
7

>100
>100
ND
ND
ND
ND
8
9
3.37
5.45
>100
>100
>100
>100
10
11
11.12
0.62
>100
>100
>100
>100
12
-
PLHSpT(1)
a IC50 Values represent the mean of at least two independent determinations.
N.D.: Not Determined
2.2. Structural and energetic analyses of the potential inhibitors
Routine methods of Molecular docking (Autodock 4.2) [24] and MM/GBSA binding
free energy calculation were used for the analyses [25-30]. As shown in Table 2,
compound 10 exhibits the strongest predicted binding affinity among the four
investigated compounds, which is consistent with the experimental data. Compound 9

fails in the docking simulation because of the neighborhood substitution of the
phosphate-containing tail, which may markedly affect the binding orientation of the
compound. Nevertheless, all the other compounds show a similar binding pose in Plk1
with the phosphate-containing tail toward K540 to match the electrostatic
environment (Fig. 3 A~C), which is consistent with the binding mode of the
co-crystallized ligands in 4WHH (Fig.3D) and 3RQ7 (Fig.3F).
Table 2. Predicted binding affinity of four investigated compounds.
compound docking score (kcal/mol) MM/GBSA (kcal/mol) IC50 (μM)
-9.69
-8.97
-9.30
N.A.
-52.47
-46.17
-39.62
N.A.
3.37
>100
>100
>100
10
6
8
9
To investigate the binding affinity difference between compounds 10 and 6/8, we
decomposed the total binding free energy into residue-inhibitor pairs [31-33]. Fig.3E
and Fig.3G shows the energetic difference spectrums of compounds 10&6 and 10&8,
respectively, where a positive value means attenuated interaction of the corresponding
residue to compound 6/8 compared with compound 10. Compared with compounds 6
and 8, compound 10 shows a 180º-rotated phenylmethanol-containing head (Fig.3A),
which forms strong interaction with R516 and F535 (Fig.3E and Fig.3G). One may
concern that the docking pose of the phenylmethanol-containing head in compound
10 cannot overlap well with the co-crystallized ligand in 4WHH (Fig.3D), which
contains a similar group as compound 10. This is because the modified backbone of
compound 10 hinders the phenylmethanol-containing head staying in the original

place (red box in Fig.3D). Nevertheless, the new binding position of the
phenylmethanol-containing head is similar with another co-crystallized ligand in
3RQ7 (red box in Fig.3F), indicating that the predicted binding mode of compound
10 is reasonable for analysis. Besides the weakened interaction with R516 and F535
of compounds 6 and 8, the benzoimidazole-containing tail of compound 6 may be too
rigid to adopt a suitable binding conformation in Plk1, which also leads to the
attenuated interaction with N533 and K540 (Fig.3E) compared with compound 10;
and the meta substitution of the phosphate-containing tail to benzene in compound 8
may modify the orientation of the backbone of the compound and thus leads to the
decreased interaction with Y481 and F482 (Fig.3C).

Fig.3. Binding modes and energetic analyses of compounds 10 (A), 6 (B) and 8 (C). Binding
energy difference based on residue-inhibitor pairs for compounds 6&10 and 8&10 were shown in
panel E and G, respectively (calculated by ΔΔG=ΔG6/8−ΔG10), where a positive value means
attenuated binding affinity of the corresponding residue to compound 6/8 compared with
compound 10. Here, vital residues contributing to the energetic difference were colored in yellow
stick model in panels A~C. To validate the docking pose, compound 10 was overlapped with the
co-crystallized ligands in 4WHH (gray in panel D) and 3RQ7 (green in panel F).
3. Conclusion
In summary, we have designed and synthesized a new series of non-peptide inhibitors

targeting the polo-box domain of Polo-like kinase 1 based on the reported tripeptide
Plk1 PBD inhibitor. All of the synthesized compounds were evaluated for their in
vitro Plk1 PBD inhibitory activity. Among them, compounds 10, 11 and 12 containing
the 1,4-regioisomer triazolyl group were significantly active with IC50 values ranging
from 3.37 to 11.12 μM. Molecule docking studies were performed and suggested
possible binding orientation, MM/GBSA binding free energy calculation were in
agreement with the observed experimental results.
Comparing to the reported phosphate peptides and peptidomimetics, our compounds
showed moderate activity to Plk1 PBD. However, the non-peptide skeleton may have
advantage over other peptide based inhibitors. The newly identified compounds
represent a novel non-peptide Plk1 PBD inhibitors with high selectivity to Plk2 PBD
and Plk3 PBD. These preliminary research findings provided novel lead compounds
for further optimization towards the discovery and development of novel non-peptide
Plk1 PBD inhibitors as potential anticancer agents.
4. Experimental section
4.1 Chemistry.
All reagents were purchased from commercial sources. Organic solutions were
concentrated in a rotary evaporator (Büchi Rotavapor) below 50 °C under reduced
pressure. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm
1
13
silica gel plates(GF-2.5) and visualized under UV light. The H NMR and C NMR
spectra were measured on a Bruker AV-300 instrument using deuterated solvents with

tetramethylsilane (TMS) as internal standard. ESI-MS and high-resolution mass
spectra (HRMS) were recorded on a Water Q-Tof micro mass spectrometer.
4.1.1. (8-azidooctyl)benzene (14)
8-Phenyl-1-octanol (13) (0.50 g, 2.42 mmol) was dissolved in dichloromethane (10
ml), triethylamine (0.49 mg, 4.85 mmol) was added. Methanesulfonyl chloride (0.42 g,
3.63 mmol) was added dropwise at 0-5 ℃. The mixture was stirred at room
temperature for 2 hrs. Then dichloromethane (20 ml) was added and the mixture was
washed with water, dried, evaporated and used without further purification. The
residue was then dissolved in DMF (10ml), NaN3 (0.47 g, 7.27 mmol) was added and
the mixture was stirred at 80℃ for 5 hrs. Water (100ml) was added and the mixture
was extracted by ethyl acetate (50 ml*3). The organic phase was collected, dried,
evaporated and the purified by column chromatography (PE). 14 was obtained as a
1
colorless oil (0.45 g, yield 80.3% for two steps). H NMR (300 MHz, CDCl3) δ:
7.60-6.89 (m, 5H), 3.31 (t, J = 6.9 Hz, 2H), 2.70 (t, J = 6.9 Hz, 2H), 1.80-1.56 (m,
4H), 1.41 (s, 8H) ppm.
4.1.2. Benzyl (S)-2-(Boc-amino)pent-4-ynoate (16)
(S)-N-Boc-Propargyl glycine (15) (0.80 g, 3.75 mmol) was dissolved in DMF (5 ml)，
K2CO3 (1.04 g, 7.50 mmol) was added. Then benzyl bromide (0.40 ml, 3.38 mmol)
was added and stirred at room temperature for 1 hr. Water (50ml) was added and the
mixture was extracted by ethyl acetate (30 ml*3), the organic phase was collected,

dried, evaporated and the purified by column chromatography (PE:EA=10:1). 16 was
1
obtained as a colorless oil (0.89 g, yield 78.2%). H NMR (300 MHz, CDCl3) δ: 7.35
(s, 5H), 5.38 (d, J = 7.2 Hz, 1H), 5.29-5.07 (m, 2H), 4.58-4.40 (m, 1H), 2.82-2.59 (m,
2H), 2.01 (s, 1H), 1.44 (s, 9H) ppm; HRMS (ESI-TOF) m/z calc’d for C17H21NO4
[M+H]+ 304.1471, found 304.1815.
4.1.3. (S)-2-(N-Boc)-3-(1-(8-phenylhexyl)-1H-1,2,3-triazol-5-yl)alanine benzyl ester
(17)
14 (2.52 g, 10.88 mmol), 16 (3.30 g, 10.88 mmol) and catalytic amount of
Cp*RuCl(PPh3)2 was dissolved in dichloromethane (50 ml). The mixture was heated
to reflux for 12 hrs and then purified by column chromatography (PE:EA=2:1). 17
1
was obtained as a colorless oil (3.00 g, yield 63.5%). H NMR (300 MHz, CDCl3) δ:
7.42-7.08 (m, 11H), 5.67-5.51 (m, 1H), 5.24-4.99 (m, 2H), 4.58 (d, J = 5.3 Hz, 1H),
4.13 (dd, J = 13.0, 6.5 Hz, 2H), 3.28-3.04 (m, 2H), 2.57 (t, J = 7.6 Hz, 2H), 1.88-1.66
(m, 2H), 1.66-1.50 (m, 2H), 1.40 (s, 9H), 1.34-1.15 (m, 8H) ppm; HRMS (ESI-TOF)
m/z calc’d for C31H42N4O4 [M+H]+ 535.3206, found 535.3320.
4.1.4. Methyl 3-(4-((ethoxymethoxy)methyl)benzamido)propanoate (20)
4-((Ethoxymethoxy)methyl)benzoic acid (18) (0.67 g, 3.19 mmol), HOBt (0.43 g,
3.19 mmol), EDCI(0.60 g, 3.19 mmol) was mixed with dichloromethane (20 ml).
DIPEA(1.29 g, 12.75 mmol) dissolved in dichloromethane (10 ml) was added in drop,
the mixture was stirred for 0.5 hr and then methyl 3-amino propanoate(0.33 g, 3.19

mmol) was added. The mixture was stirred at room temperature for 5 hrs and then
washed using 1 N HCl (10ml*2) followed by water (10ml*2). The organic phase was
dried, concentrated and purified by column chromatography (PE:EA=2:1). 20 was
1
obtained as a colorless oil 0.80 g, yield 85.0%). H NMR (300 MHz, CDCl3) δ: 7.76
(d, J = 7.3 Hz, 2H), 7.41 (d, J = 7.4 Hz, 2H), 6.97 (s, 1H), 4.76 (s, 2H), 4.64 (s, 2H),
3.83-3.52 (m, 7H), 2.66 (t, J = 5.4 Hz, 2H), 1.23 (t, J = 7.0 Hz, 3H) ppm; HRMS
(ESI-TOF) m/z calc’d for C15H21NO5 [M+H]+ 296.1420, found 296.1505.
4.1.5.
Benzyl
(S)-2-(3-(4-((ethoxymethoxy)methyl)benzamido)propanamido)-3-(1-(8-phenyloctyl)-1
H-1,2,3-triazol-4-yl)propanoate (21)
20 (0.38 g, 1.35 mmol) was dissolved in 1,4-dioxane (10ml), 3N NaOH a.q. (10ml)
was added. The mixture was stirred at room temperature for 2 hrs. The solution was
extracted by ethyl acetate (20 ml*3). The organic phase was collected, dried,
evaporated to get the corresponding acid. The acid, HOBt (0.18 g, 1.35 mmol),
EDCI(0.26 g, 1.35 mmol) was mixed with dichloromethane (10 ml). DIPEA(0.58 g,
4.49 mmol) dissolved in dichloromethane (5 ml) was added in drop, the mixture(A)
was
stirred
for
0.5
hr.
At
the
same
time,
(S)-2-(N-Boc)-3-(1-(8-phenylhexyl)-1H-1,2,3-triazol-5-yl)alanine benzyl ester (17)
(0.33 g, 3.19 mmol) was dissolved in dichloromethane (10 ml), trifluoroacetic acid (1
ml) was added. The mixture was stirred at room temperature for 0.5 hr and evaporated
to dry. Then saturated NaHCO3 solution (10 ml) was added and extracted using

dichloromethane (10 ml*3). The organic phase was collected, dried and contracted
and then mixture(A) was added in. After stirred at room temperature for 5 hrs, the
solution was diluted using dichloromethane (20 ml), washed by 1 N HCl (10 ml*2),
saturated NaHCO3 solution (10 ml*2) and water (10 ml*2). The organic layer was
purified by column chromatography (PE:EA=2:1). 21 was obtained as a colorless oil
1
(0.78 g, yield 87.0%). H NMR (300 MHz, CDCl3) δ: 7.82-7.55 (m, 3H), 7.40-7.06
(m, 14H), 5.24-5.03 (m, 2H), 4.85 (dd, J = 13.3, 6.4 Hz, 1H), 4.78 (s, 2H), 4.61 (s,
2H), 4.18-3.99 (m, 2H), 3.72-3.53 (m, 4H), 3.26-3.03 (m, 2H), 2.55 (dd, J = 15.5, 7.5
Hz, 4H), 1.80-1.65 (m, 2H), 1.65-1.46 (m, 2H), 1.33-1.09 (m, 11H) ppm; HRMS
(ESI-TOF) m/z calc’d for C40H51N5O6 [M+H]+ 698.3839, found 698.3995.
4.1.6.
(S)-2-(3-(4-((ethoxymethoxy)methyl)benzamido)propanamido)-3-(1-(8-phenyloctyl)-1
H-1,2,3-triazol-4-yl)propanoic acid (22)
21 was dissolved in methanol, 10%Pd-C (catalytic amount) was added in. The
mixture was hydrogenated at normal pressure for 40 min. The insoluble was filtered
off and the solvent was removed under vacuum and the residue was used directly for
the next step without further purification.
4.1.7. Diethyl (4-amino-3-nitrophenethyl)phosphonate (25)
2-(4-Aminophenyl)ethan-1-ol (23) (3.00 g, 21.87 mmol) was dissolved in toluene (50
ml). Acetic anhydride (5.6 g, 54.67 mmol), triethylamine (11.06 g, 109.34 mmol) was

added. The mixture was heated to 80℃ and stirred for 1 hr. The solution was then
washed using water (50ml*3), dried and evaporated to dry. Then the residue was
dissolved in dichloromethane (50 ml), nitric acid (1.60 ml, 35.43 mmol) was added at
0 ℃, the mixture was stirred at 0 ℃ for 1 hr and then poured into crashed ice (50 g).
The mixture was extracted using dichloromethane (50 ml*3). The organic layer was
collected, dried and evaporated to get yellow oil. The yellow oil was then added to 10
N NaOH (30ml), and stirred at room temperature for 15 min. The mixture was
extracted by ethyl acetate (30 ml*3). The organic phase was collected, dried,
evaporated to dry. The residue was mixed with 45% HBr (40 ml), and heated to reflux
for 10 hrs. After cooled to room temperature, the mixture was extracted by ethyl
acetate (30 ml*3), washed by water (10 ml*2) and then saturated NaHCO3 solution
(10 ml*2). The organic layer was dried to get yellow oil and used for next step
without further purification. The yellow oil was dissolved in triethyl phosphite (8.14 g,
48.96 mmol), and the mixture was stirred at 120℃ for 5 hrs. The target compound
(25) was purified by column chromatography (EA) as a yellow oil (4.00g, yield
1
81.1%). H NMR (300 MHz, CDCl3) δ: 7.91 (d, J = 1.0 Hz, 1H), 7.33-7.05 (m, 1H),
6.80 (d, J = 8.6 Hz, 1H), 6.23 (s, 2H), 4.28-3.84 (m, 4H), 2.97-2.66 (m, 2H),
2.26-1.85 (m, 2H), 1.31 (t, J = 7.1 Hz, 6H) ppm.
4.1.8. Diethyl (3,4-diaminophenethyl)phosphonate (26)
25 (2.00g) was dissolved in methanol (30 ml), 10%Pd-C (0.05 g) was added in. The
mixture was hydrogenated at normal pressure for 4 hrs. The insoluble was filtered off

and the solvent was removed to gain 26 as a brown oil (1.80 g, yield 91.6%). 1H NMR
(300 MHz, CDCl3) δ: 6.67-6.55 (m, 1H), 6.53-6.44 (m, 2H), 4.19-3.94 (m, 4H), 3.52
(s, 4H), 2.83-2.64 (m, 2H), 2.10-1.82 (m, 2H), 1.30 (t, J = 7.1 Hz, 6H) ppm.
4.1.9. Dibenzyl (3-(N-Boc-amino)phenethyl)phosphonate (28a)
2-(3-Aminophenyl)ethan-1-ol (27a) (5.00 g, 36.46 mmol) was dissolved in
1,4-dioxane (250 ml), Boc2O(7.96 g, 36.46 mmol) was added. The mixture was stirred
at room temperature for 10 hrs and then extracted by ethyl acetate (250 ml*3), washed
by water (250 ml*2) and then saturated NaHCO3 solution (250 ml*2). The organic
layer was dried and concentrated to get pale yellow oil. The pale yellow oil was then
dissolved in dichloromethane (200 ml), triethylamine (6.94 g, 68.61 mmol) was added
and the mixture stirred with ice bath for 10 min. Methanesulfonyl chloride (5 ml,
51.45 mmol) was added in drop and then stirred at room temperature for 2 hrs. The
mixture was washed by water (100 ml*2). The organic layer was dried and evaporated
to get yellow oil and used for next step without further purification. The yellow oil
was dissolved in DMF (40 ml), NaBr (14.12 g, 137.21 mmol) and catalytic amount of
TBAB was added. The mixture was stirred at room temperature for 36 hrs. Water (400
ml) was added and the mixture was extracted by ethyl acetate (250 ml*3). The organic
layer was collected and washed by water (250 ml*2), dried and the solvent removed
to get white solid. The white solid (1.42 g, 4.66 mmol), TBAI(5.13 g, 13.99 mmol),
Cs2CO3(4.56 g, 13.99 mmol) and dibenzyl phosphite (1.47 g, 5.60 mmol) were mixed
with dry DMF (5 ml). The mixture was stirred at room temperature for 10 hrs and the

water (50 ml) added, extracted by ethyl acetate (30 ml*3). The organic layer was
collected and washed by water (50 ml*2), dried and purified by column
chromatography (PE:EA=2:1) as a white powder (28a) (4.97 g, yield 43.0% for four
1
steps). H NMR (300 MHz, CDCl3) δ: 7.44-7.17 (m, 13H), 7.00 (d, J = 8.3 Hz, 2H),
5.20-4.89 (m, 4H), 2.82 (dd, J = 16.9, 10.1 Hz, 2H), 2.11-1.87 (m, 2H), 1.52 (s, 9H)
ppm.
4.1.10. Dibenzyl (3-(N-Boc-aminomethyl)phenethyl)phosphonate (28b)
28b was prepared under similar conditions as described for 28a (34.0 % yield for four
steps). 1H NMR (300 MHz, CDCl3) δ: 7.51-6.88 (m, 14H), 5.02-4.74 (m, 5H), 4.18 (d,
J = 5.7 Hz, 2H), 2.88-2.64 (m, 2H), 2.05-1.80 (m, 2H), 1.39 (s, 9H) ppm; HRMS
(ESI-TOF) m/z calc’d for C28H34NO5P [M+H]+ 496.2175, found 496.2522.
4.1.11. Dibenzyl (3-aminophenethyl)phosphonate (29a)
28a was dissolved in dichloromethane (10 ml), trifluoroacetic acid (1 ml) was added.
The mixture was stirred at room temperature for 0.5 hr and evaporated to dry. Then
saturated NaHCO3 solution (10 ml) was added and extracted using dichloromethane
(10 ml*3). The organic phase was collected, dried and evaporated to get 29a as a
yellow oil and used directly for next step without further purification.
4.1.12. Dibenzyl (3-aminomethylphenethyl)phosphonate (29b)
29b was prepared under similar conditions as described for 29a and used directly for

next step without further purification.
4.1.13. ((2-Azidoethoxy)methyl)benzene (31a)
Ethylene glycol (30a) (7.36 g, 116.93 mmol) was dissolved in DMF (100 ml), NaH
(2.34 g, 58.47 mmol) was added in part with ice bath. After 0.5 hr, benzyl bromide
(3.47 ml, 29.23 mmol) was added slowly and the mixture was stirred at room
temperature for 2 hrs. Water (500 ml) was added and the mixture was extracted by
ethyl acetate (300 ml*3). The organic layer was collected, dried and evaporated. The
residue (3.00 g, 19.71 mmol) and dissolved in dichloromethane (50 ml), triethylamine
(3.99 g, 39.42 mmol) was added and the mixture was stirred with ice bath for 5 min.
Methanesulfonyl chloride (3.39 g, 29.57 mmol) was added in drop and then stirred at
room temperature for 1 hrs. The mixture was washed by water (30 ml*2). The organic
layer was dried and evaporated to get yellow oil. The yellow oil was dissolved in
DMF (20 ml), azidotrimethylsilane (4.55 g, 39.42 mmol) and K2CO3(7.27 g, 15.49
mmol) were added. The mixture was stirred at 100℃ for 5 hrs. Water (100 ml) was
added and the mixture was extracted by ethyl acetate (80 ml*3). The organic layer
was collected and washed by water (100 ml*2) and dried. 31a was purified by column
chromatography (PE:EA=20:1) as a colorless oil (2.60g, yield 76.4% for three steps).
1H NMR (300 MHz, CDCl3) δ: 8.09-7.96 (m, 2H), 7.56-7.46 (m, 1H), 7.44-7.35 (m,
2H), 4.60 (s, 2H), 4.36 (t, J = 6.2 Hz, 2H), 3.41 (t, J = 6.7 Hz, 2H) ppm.
4.1.14. ((3-Azidopropoxy)methyl)benzene (31b)

31b was prepared under similar conditions as described for 31a (88.1% yield for three
steps). 1H NMR (300 MHz, CDCl3) δ: 8.13-7.90 (m, 2H), 7.65-7.34 (m, 3H), 4.42 (t, J
= 6.1 Hz, 2H), 3.48 (t, J = 6.7 Hz, 2H), 2.17-1.90 (m, 2H) ppm.
4.1.15. ((4-azidobutoxy)methyl)benzene (31c)
31c was prepared under similar conditions as described for 31a (80.1% yield for three
steps). 1H NMR (300 MHz, CDCl3) δ: 7.45-7.23 (m, 5H), 4.56 (s, 2H), 3.63-3.49 (m,
2H), 3.42-3.23 (m, 2H), 1.87-1.67 (m, 4H) ppm.
4.1.16. 2-(4-(N-Boc-aminomethyl)-1H- 1,2,3-triazol-1-yl)ethyl benzyl ether (32a)
N-Boc-2-Propynylamine (0.45 g, 2.88 mmol), 31a (0.59 g, 2.88 mmol) was
dissolved in tert-butanol (5 ml). Catalytic amount of CuSO4 was dissolved in water (5
ml), Vc-Na was added to change the color from blue to yellow. Then the solution was
added to the tert-butanol solution and stirred for 2 hrs. 32a was purified by column
1
chromatography (PE:EA=1:1) as a pale yellow oil (0.82g, yield 79.8 %). H NMR
(300 MHz, CDCl3) δ: 7.63 (s, 1H), 7.36-7.19 (m, 5H), 5.29 (s, 1H), 4.58-4.45 (m, 4H),
4.38 (m, 2H), 3.80 (t, J = 5.0 Hz, 2H), 1.42 (s, 9H) ppm; HRMS (ESI-TOF) m/z
calc’d for C17H24N4O3 [M+H]+ 333.1848, found 333.1938.
4.1.17. 3-(4-(N-Boc-aminomethyl)-1H- 1,2,3-triazol-1-yl)propyl benzyl ether (32b)
1
32b was prepared under similar conditions as described for 32a (yield 86.9%). H
NMR (300 MHz, CDCl3) δ: 7.95-7.84 (m, 2H), 7.59-7.31 (m, 4H), 5.47 (s, 1H),

4.54-4.38 (m, 2H), 4.33-4.21 (m, 4H), 2.37-2.23 (m, 2H), 1.34 (s, 9H) ppm; HRMS
(ESI-TOF) m/z calc’d for C18H24N4O4 [M+H]+ 361.1798, found 361.1892.
4.1.18. 4-(4-(N-Boc-aminomethyl)-1H- 1,2,3-triazol-1-yl)butyl benzyl ether (32c)
1
32c was prepared under similar conditions as described for 32a (yield 80.3%). H
NMR (300 MHz, CDCl3) δ: 7.50 (s, 1H), 7.33 (m, 5H), 5.27 (s, 1H), 4.49 (s, 2H),
4.43-4.29 (m, 4H), 3.49 (t, J = 6.0 Hz, 2H), 2.11-1.87 (m, 2H), 1.77-1.57 (m, 2H),
1.44 (s, 9H) ppm; HRMS (ESI-TOF) m/z calc’d for C19H28N4O3 [M+H]+ 361.2161,
found 361.2263.
4.1.19. Dibenzyl (2-(4-(N-Boc-aminomethyl)-1H-1,2,3-triazol-1-yl)ethyl)phosphonate
(33a)
1
33a was prepared under similar conditions as described for 28a (yield 79.0%). H
NMR (300 MHz, CDCl3) δ: 7.53-7.11 (m, 11H), 5.21-4.80 (m, 5H), 4.54-4.17 (m, 4H),
2.50-2.24 (m, 2H), 1.41 (s, 9H) ppm; HRMS (ESI-TOF) m/z calc’d for C25H33N4O5P
[M+H]+ 487.2032, found 487.2118.
4.1.20.
Dibenzyl
(3-(4-(N-Boc-aminomethyl)-1H-1,2,3-triazol-1-yl)propyl)
phosphonate (33b)
1
33b was prepared under similar conditions as described for 28a (yield 50.4%). H
NMR (300 MHz, CDCl3) δ: 7.72-7.20 (m, 11H), 5.28 (s, 1H), 5.09-4.91 (m, 4H),
4.43-4.24 (m, 4H), 2.21-2.02 (m, 2H), 1.79-1.62 (m, 2H), 1.45 (s, 9H) ppm; HRMS

(ESI-TOF) m/z calc’d for C25H33N4O5P [M+H]+ 501.2189, found 501.2275.
4.1.21. Dibenzyl (4-(4-(N-Boc-aminomethyl)-1H-1,2,3-triazol-1-yl)butyl)phosphonate
(33c)
1
33c was prepared under similar conditions as described for 28a (yield 74.5%). H
NMR (300 MHz, CDCl3) δ: 7.72-7.20 (m, 11H), 5.42 (s, 1H), 5.08-4.77 (m, 4H),
4.40-4.06 (m, 4H), 2.48-2.20 (m, 1H), 1.80-1.47 (m, 2H), 1.39 (s, 9H), 0.93 (d, J =
5.8 Hz, 3H) ppm; HRMS (ESI-TOF) m/z calc’d for C26H35N4O5P [M+H]+ 515.2345,
found 515.2448.
4.1.22. Dibenzyl (2-(4-aminomethyl-1H-1,2,3-triazol-1-yl)ethyl)phosphonate (34a)
34a was prepared under similar conditions as described for 29a and used directly for
next step without further purification.
4.1.23. Dibenzyl (3-(4-aminomethyl-1H-1,2,3-triazol-1-yl)propyl)phosphonate (34b)
34b was prepared under similar conditions as described for 29a and used directly for
next step without further purification.
4.1.24. Dibenzyl (4-(4-aminomethyl-1H-1,2,3-triazol-1-yl)butyl)phosphonate (34c)
34c was prepared under similar conditions as described for 29a and used directly for
next step without further purification.

4.1.25. 2-(5-(N-Boc-aminomethyl)-1H- 1,2,3-triazol-1-yl)ethyl benzyl ether (35)
N-Boc-2-Propynylamine (0.45 g, 2.88 mmol), 31a (0.59 g, 2.88 mmol) and catalytic
amount of Cp*RuCl(PPh3)2 was dissolved in DCE (5 ml). The mixture was heated to
reflux and stirred for 10 hrs and 35 was purified by column chromatography
(PE:EA=1:1) as a pale yellow oil (0.57g, yield 55.2 %). 1H NMR (300 MHz, CDCl3)
δ: 7.49 (d, J = 12.0 Hz, 1H), 7.27-7.00 (m, 5H), 5.25 (s, 1H), 4.50-4.17 (m, 6H),
3.82-3.65 (m, 2H), 1.28 (s, 9H) ppm; HRMS (ESI-TOF) m/z calc’d for C17H24N4O3
[M+H]+ 333.1848, found 333.1936.
4.1.26. Dibenzyl (2-(5-(N-Boc-aminomethyl)-1H-1,2,3-triazol-1-yl)ethyl)phosphonate
(36)
1
36 was prepared under similar conditions as described for 28a (yield 64.5%). H
NMR (300 MHz, CDCl3) δ: 7.30-7.15 (m, 11H), 4.95-4.75 (m, 5H), 4.43-4.24 (m,
2H), 4.20-4.11 (m, 2H), 2.34-2.18 (m, 2H), 1.41 (s, 9H) ppm; HRMS (ESI-TOF) m/z
calc’d for C25H33N4O5P [M+H]+ 487.2032, found 487.2105.
4.1.27. Dibenzyl (2-(5-aminomethyl-1H-1,2,3-triazol-1-yl)ethyl)phosphonate (37)
37 was prepared under similar conditions as described for 29a and used directly for
next step without further purification.
4.1.28.
(S)-(2-(2-(1-(3-(4-(hydroxymethyl)benzamido)propanamido)-2-(1-(8-phenyloctyl)-1H-

1,2,3-triazol-5-yl)ethyl)-1H-benzo[d]imidazol-6-yl)ethyl)phosphonic acid (6)
22 (0.26 g, 0.37 mmol), HATU(0.12 g, 0.37 mmol), DIPEA(0.14 g,1.10 mmol) were
mixed with CH2Cl2 (10 ml). After stirred with ice bath for 0.5 h, 26 (0.10 g, 0.37
mmol) was added in and the mixture was stirred at room temperature for 3 hrs. The
solution was washed by water (10 ml*2), dried and purified by column
chromatography (PE:EA=20:1). The colorless oil gained was then dissolved in acetic
anhydride (5 ml) and the mixture was stirred at 65 ℃ for 6 hrs. Then saturated
NaHCO3 solution (20 ml) was added and the mixture was extracted by CH2Cl2 (20
ml*3), the organic layer was collected and the solvent was removed under vacuum
and the residue was then dissolved in CH2Cl2 (1 ml). Bromotrimethylsilane (2 ml)
was added to the solution and the mixture was stirred at room temperature for 10 hrs.
Methanol (10 ml) was added and stirred at room temperature for more 0.5 hr. 6 was
purified by the preparative RP-HPLC (XBridgeTM , Prep C18, 50 × 250 mm, 10 μm)
using an appropriate water/acetonitrile gradient in the presence of 0.05% TFA
1
(10.0mg, yield 3.7% for three steps). H NMR (300 MHz, MeOD) δ: 7.80-7.03 (m,
13H), 5.53-5.37 (m, 1H)，4.62 (s, 2H), 4.25 (t, J = 6.9 Hz, 2H), 3.72-3.46 (m, 4H),
3.03 (s, 2H), 2.63-2.50 (m, 4H), 2.07-1.90 (m, 2H), 1.80-1.68 (m, 2H), 1.58-1.46 (m,
13
2H), 1.27-1.07 (m, 8H) ppm; C NMR (75 MHz, MeOD) δ: 173.3, 167.2, 147.5,
146.0, 142.1, 141.2, 138.5, 136.0, 134.7, 133.3, 131.0, 129.8, 128.6, 128.1, 127.7,
1255, 124.3, 115.0, 114.2, 64.7, 51.5, 49.9, 37.3, 36.2, 35.8, 35.3, 34.6, 31.2, 29.8,
29.2, 28.7, 27.4, 27.1, 20.8 ppm; HRMS (ESI-TOF) m/z calc’d for C38H48N7O6P
[M+H]+ 730.3407, found 730.3463.