Novel leucine ureido derivatives as aminopeptidase N inhibitors using click chemistry

Article abstract of DOI:10.1016/j.bmc.2018.04.041

The over-expression of aminopeptidase N on diverse malignant cells is associated with the tumor angiogenesis and metastasis. In this report, one new series of leucine ureido derivatives containing the triazole moiety was designed, synthesized and evaluated as APN inhibitors. Among them, compound 13v showed the best APN inhibition with an IC₅₀ value of 0.089 ± 0.007 μM, which was two orders of magnitude lower than that of bestatin (IC₅₀ = 9.4 ± 0.5 μM). Compound 13v also showed dose-dependent anti-angiogenesis activities. Even at the lower concentration (10 μM), compound 13v presented similar anti-angiogenesis activity compared with bestatin at 100 μM in both the human umbilical vein endothelial cells (HUVECs) capillary tube formation assay and the rat thoracic aorta rings test. Moreover, compared with bestatin, 13v exhibited comparable, if not better in vivo anti-metastasis activity in a mouse H22 pulmonary metastasis model.

Full text of DOI:10.1016/j.bmc.2018.04.041

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel leucine ureido derivatives as aminopeptidase N inhibitors using
click chemistry
Jiangying Cao a, Chunhua Ma b, Jie Zang a, Shuai Gao a, Qianwen Gao a, Xiujie Kong a, Yugang Yan a,
Xuewu Liang a, Qin’ge Ding a, Chunlong Zhao a, Binghe Wang c, Wenfang Xu a, , Yingjie Zhang a,
⇑
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China
b School of Chemistry and Chemical Engineering, Henan Normal University, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals,
Key Laboratory of Green Chemical Media and Reactions of Ministry of Education, Xinxiang 453007, China
c Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The over-expression of aminopeptidase N on diverse malignant cells is associated with the tumor angio-
genesis and metastasis. In this report, one new series of leucine ureido derivatives containing the triazole
moiety was designed, synthesized and evaluated as APN inhibitors. Among them, compound 13v showed
Received 24 January 2018
Revised 17 April 2018
Accepted 19 April 2018
Available online xxxx
the best APN inhibition with an IC50 value of 0.089 0.007
than that of bestatin (IC50 = 9.4 0.5 M). Compound 13v also showed dose-dependent anti-angiogenesis
activities. Even at the lower concentration (10 M), compound 13v presented similar anti-angiogenesis
activity compared with bestatin at 100 in both the human umbilical vein endothelial cells
lM, which was two orders of magnitude lower
l
l
Keywords:
Aminopeptidase N
CD13
Anti-angiogenesis
Anti-metastasis
Triazole
l
M
(HUVECs) capillary tube formation assay and the rat thoracic aorta rings test. Moreover, compared with
bestatin, 13v exhibited comparable, if not better in vivo anti-metastasis activity in a mouse H22 pul-
monary metastasis model.
Ó 2018 Published by Elsevier Ltd.
1. Introduction
products against APN have been documented, including amas-
tatin11, actinonin12, phebestin13, and lapstatin.14 Our group has a
Aminopeptidase N (APN, CD13) is a zinc-dependent membrane
bound exopeptidase, consisting of 967 amino acids with a short
cytoplasmic part, a single transmembrane domain and a large
extracellular catalytic domain.1 The enzyme preferentially hydro-
lyzes basic or neutral amino acid residues from the unsubstituted
N-terminals of oligopeptides, such as enkephalins, angiotensins,
P-substance, cytokines and others.2 As a widely distributed and
over-expressed enzyme in malignant cells, it is associated with
the growth, invasion and metastasis of diverse cancers.3–7 More-
over, documents suggested that APN expression, specially on the
endothelium of angiogenic vasculature, could be activated by
angiogenic signals and is essential for capillary tube formation.8,9
All these findings make APN a promising antitumor target.
long-standing interest in the development of synthetic APNIs as
antitumor agents15–21, among which the leucine ureido-based
APNIs (2, Fig. 1) exhibited promising in vitro and in vivo anti-angio-
genisis and anti-metastasis potency.18–21 Our previous structure-
activity relationship studies revealed that structural modifications
of the aryl group of 2 were well tolerated.18–21 1,2,3-triazole, which
could be readily obtained by click chemistry, is widely used in var-
ious biologically active compounds due to its desirable features in
the context of medicinal chemistry.22–26 For example, triazoles are
stable to acid and basic hydrolysis, redox conditions and metabolic
degradation. This heterocycle also has the potential to form hydro-
gen bonds as well as
p stacking interactions. Herein, the 1,2,3-tri-
azole moiety was introduced as the surrogate for the aryl group in
compound 2 to get a novel series of leucine ureido-based APNIs (3,
Fig. 1). All target compounds were synthesized efficiently using
click chemistry and all the leucine ureido-based compounds exhib-
ited more potent APN inhibitory activities than the positive control
bestatin. The superior APN inhibitory activities of several represen-
tative compounds were validated using cell-based APN inhibitory
assays. The most potent compound 13v exhibited promising
in vitro anti-angiogenisis potency and in vivo anti-metastasis
Based on their different sources, the APN inhibitors (APNIs) can
be divided into the natural products and synthetic compounds.
Bestatin (1, Fig. 1), isolated from the Streptomyces oliuoreticuli by
Umezawa et al., is the first reported and only marketed natural
dipeptidomimetic inhibitor of APN.10 After that, many natural
⇑
Corresponding authors.
E-mail address: zhangyingjie@sdu.edu.cn (Y. Zhang).
https://doi.org/10.1016/j.bmc.2018.04.041
0968-0896/Ó 2018 Published by Elsevier Ltd.
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2
J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3. Results
The newly synthesized leucine ureido derivatives were firstly
evaluated for their enzymatic inhibition using porcine kidney
APN (Microsomal, Biocol) with bestatin as the positive control
(Table 1). One isoleucine ureido derivative 14 and phenylglycine
ureido derivative 15 were also synthesized and evaluated, which
exhibited much less potent APN inhibition than their correspond-
ing leucine ureido derivatives 13a and 13d, respectively, suggest-
ing that the leucine residue is more suitable for APN recognition.
Introduction of various substituents on phenyl group of 13a led
to mono-substituted analogs 13b–13w, 20a–20b and multi-
substituted analogs 13aa–13ff. Comparing the mono-substituted
analogs, it was found that for chlorine (13b–13d), bromine (13e–13g),
methyl (13h–13j), methoxyl (13k–13m), ethyoxyl (13s–13u),
benzyloxyl (13v–13w) and amino (20a–20b) substituted analogs,
their APN inhibitory activity order is ortho-substituted > meta-
substituted > para-substituted. However, the similar trend was
not observed in nitro (13n–13p) or cyano (13q–13r) substituted
analogs. Among multi-substituted analogs 13aa–13ff, compounds
containing two ortho-substituents displayed more potent activity
than their counterparts with other substitutions patterns (13aa
vs 13dd, and 13cc vs 13ee). Moreover, compounds containing
two ortho-substituents are more potent than their counterparts
containing one single ortho-substituent (13aa vs 13b, 13bb vs
13h, and 13cc vs 13k). Replacement of the benzyl group of 13a
using other functional groups led to derivatives 25a–25h with
comparable or better APN inhibitory activities to 13a, indicating
that different substituents on the triazole were generally well
tolerated. It was worth noting that all our newly synthesized leu-
cine ureido derivatives exhibited better APN inhibitory potency
than the positive control bestatin. The most potent compound
Fig. 1. The design of leucine ureido-based triazole derivatives as APN inhibitors.
potency. Finally, the molecular docking study was used to postu-
late the binding mode of 13v in the active site of APN.
2. Chemistry
The target compounds were efficiently synthesized following
the procedures shown in the Schemes 1–4.
As shown in Scheme 1, compounds 4a–4c reacted with triphos-
gene to yield isocyanate, which was then immediately reacted with
propargylamine to generate the ureido derivatives 5a-5c. The
ureido derivatives 5a-5c were converted into key intermediates
hydroxamic acids 6a-6c in the presence of NH2OK in dry methanol.
As shown in Scheme 2, the aldehydes (7a–7u, 7aa–7ff, 9v–9w)
were converted into their alcohol derivatives (8a–8u, 8aa–8ff,
10v–10w) via reduction by sodium borohydride in methanol. Com-
pounds 9v–9w were generated by reaction of 7v–7w with benzyl
bromide. The hydroxyl group of compounds 8a–8u, 8aa–8ff and
10v–10w were converted into a mesyl group under basic condi-
tions and then reacted with sodium azide to yield the azide deriva-
tives (12a–12w, 12aa–12ff). The target compounds 13a-13w and
13aa-13ff were prepared by coupling 6a with compounds 12a–
12w and 12aa–12ff via click chemistry, respectively. Compounds
14 and 15 were prepared by coupling 6b with 12a and 6c with
12d via click chemistry, respectively.
13v (IC50 = 0.089 0.007
lM) was over 100-fold more potent than
bestatin (IC50 = 9.4 0.5
lM).
Furthermore, some compounds with potent enzyme inhibition
activity were selected to evaluate their inhibitory capacities
against human APN stemming from ES-2 and PLC/PRF/5 cell lines.
The results in Table 2 show that compound 13v was still the most
potent compound with IC50 values over an order of magnitude
lower than that of bestatin.
MTT assay was performed to evaluate the in vitro cytotoxicity of
compounds 13v and 13w. The results listed in Table 3 show that
both 13v and 13w presented better proliferation inhibition than
bestatin against all the cell lines tested.
Compound 13v was further progressed to human umbilical vein
endothelial cells (HUVECs) tubular structure formation assay to
evaluate its anti-angiogenesis activity. The results in Fig. 2 show
that, compound 13v could reduce the capillary tube formation in
Scheme 3 presented the synthesis of compounds 20a–20b.
Compounds 16a–16b underwent reduction by sodium borohy-
dride to yield compounds 17a–17b, of which the nitro groups were
reduced to amino groups with sodium sulfide. Subsequently, acti-
vation of the hydroxyl group with triphenylphosphine in CCl4/DMF
and then reaction with sodium azide led to the azide derivatives
19a–19b. Compounds 19a-19b were coupled with 6a via click
chemistry to generate compounds 20a–20b.
a dose-dependent manner. It was worth noting that 10
13v presented similar anti-angiogenesis activity with 100
bestatin.
It is considered that the rat aortic ring model could simulate the
in vivo angiogenesis environment better than the HUVECs tube for-
mation model. Our results in Fig. 3 show that compound 13v could
potently reduce micro-vessel growth in a dose-dependent manner.
Moreover, similar with the results in HUVECs tubular structure for-
l
l
M of
M of
The reactions in Scheme 4 are similar to that of Scheme 2. The
aldehydes and ketones (21a–21h) underwent reduction to yield
the corresponding alcohols (22a–22h). Activation of the hydroxyl
group followed by nucleophilic substitution led to azide deriva-
tives 24a–24h, which were coupled with 6a via click chemistry
to yield compounds 25a–25h, respectively.
mation assay, 10
micro-vessel growth inhibition relative to 100
l
M of compound 13v demonstrated comparable
M of bestatin.
l
Based on the significant roles of APN in tumor metastasis5–7, we
used the mouse H22 hepatoma cell pulmonary metastasis model to
evaluate the in vivo anti-metastasis activity of 13v. The number of
metastasis nodes on the surface of pulmonary lobes was counted
to evaluate the anti-metastasis effect of tested compounds. As
depicted in Fig. 4, the number of nodes in mice treated by 13v
(60 mg/kg, ip) was lower than that of mice treated by bestatin
Scheme 1. Reagents and conditions: (a) triphosgene, NaHCO3, DCM/H2O, ice-bath,
1.5 h; (b) propargylamine, TEA, DCM, 25 °C, 12 h; (c) NH2OK, MeOH, 25 °C, 0.5 h.
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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Scheme 2. Reagents and conditions: (a) NaBH4, MeOH, ice-bath, 1 h; (b) benzyl bromide, K2CO3, DMF, 80 °C, 5 h; (c) CH3SO2Cl, DCM, TEA, ice-bath, 12 h; (d) NaN3, DMF, 25 °C,
12 h; (e) 6a for 12a-12w and 12aa-12ff, CuSO4ꢀ5H2O, sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h; (f) 6b for 12a, CuSO4ꢀ5H2O, sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2
h; (g) 6c for 12d, CuSO4ꢀ5H2O, sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h.
In order to postulate its binding mode in APN, compound 13v
was docked into the active site of APN (PDB code: 2DQM) using
Sybyl_X 1.3. As illustrated in Fig. 5A, the catalytic zinc ion of APN
0
was chelated by the hydroxamate moiety of 13v, and the S1, S1
0
and S2 pockets of APN were occupied by the leucine residue, the
two phenyl groups of 13v, respectively. Moreover, the binding
mode of 13a (Fig. 5B) was also postulated and compared to that
of 13v (Fig. 5A). It could be found that both 13a and 13v chelated
the zinc ion via the hydroxamate group and occupied the S1 pocket
via the leucine residue. Note that compound 13a could only occupy
0
the S1 pocket with its single phenyl group, while compound 13v
0
0
could occupy the S1 and S2 pockets simultaneously. This might
Scheme 3. Reagents and conditions: (a) NaBH4, MeOH, ice-bath, 1 h; (b) Na2Sꢀ9H2-
O, H2O, 100 °C, 3 h; (c) Ph3P, NaN3, DMF, CCl4, 100 °C, 12 h; (d) 6a, CuSO4ꢀ5H2O,
sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h.
rationalize the better APN inhibitory activity of 13v.
4. Conclusion
(60 mg/kg, ip). The inhibitory rates of metastasis of 13v and besta-
tin were 71% and 64%, respectively. There were no apparent loss of
body weight and no evident toxic signs in liver and spleen in the
group treated by 13v.
In summary, one novel series of triazole derivatives was
designed, synthesized and evaluated as APNIs. In the enzymatic
assay against APN, all leucine ureido-based compounds presented
much better inhibitory activities than the positive control bestatin.
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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Scheme 4. Reagents and conditions: (a) NaBH4, MeOH, ice-bath, 1 h; (b) CH3SO2Cl, DCM, TEA, ice-bath, 12 h; (c) NaN3, DMF, 25 °C, 12 h; (d) 6a, CuSO4ꢀ5H2O, sodium
ascorbate, DMSO/H2O (4:1), 25 °C, 2 h.
The best compound 13v demonstrated more potent in vitro anti-
angiogenesis activity than bestatin in both HUVECs tubular struc-
ture formation assay and rat aortic ring model. What’s more, 13v
exhibited promising anti-metastasis effects in a mouse H22 pul-
monary metastasis model, which deserve further research and
development as anti-metastatic lead compound.
DCM was concentrated under vacuum. The residue was dissolved
in EtOAc (100 mL) followed by washing with 10% HCl (3 ꢁ 100
mL), saturated NaHCO3 (3 ꢁ 100 mL) and brine (3 ꢁ 100 mL), and
drying over MgSO4 overnight. EtOAc was evaporated and the
obtained crude product was further purified by recrystallization
from ethyl acetate/petroleum ether to give compound 5a as a
white solid (1.87 g, yield: 83%), mp: 102.9–104.2 °C. 1H NMR
(300 MHz, DMSO d6): d 6.37 (d, J = 8.4 Hz, 1H), 6.25 (t, J = 5.7 Hz,
1H), 4.17 (q, J = 8.4 Hz, 1H), 3.78 (dd, J = 5.7 Hz, J = 2.4 Hz, 2H),
3.61 (s, 3H), 3.05 (t, J = 2.4 Hz, 1H), 1.68–1.54 (m, 1H), 1.47–1.42
(m, 2H), 0.89–0.84 (m, 6H); ESI-MS m/z 227.4 [M+H]+.
5. Experiment section
5.1. Chemistry: General procedures
All the materials were commercially available (Adamas-beta)
and used without further purification otherwise noted. All reac-
tions were monitored by thin-layer chromatography (TLC) on
0.25 mm silica gel plates (60 GF-254). UV light, ferric chloride
and iodine vapor were used to visualize the spots. The products
were purified by column chromatography or recrystallization.
Melting points were determined on an electrothermal melting
point apparatus without correction. ESI-MS were run on an API
4000 spectrometer. HRMS spectra were conducted by Shandong
Analysis and Test Center. 1H NMR and 13C NMR spectra were deter-
mined on a Brucker DRX spectrometer, and were described as d in
parts per million and J in Hertz, with TMS as an internal standard.
5.1.1.1. (2S,3R)-Methyl 3-methyl-2-(3-(prop-2-yn-1-yl)ureido)pen-
tanoate (5b). White solid, yield: 81%, mp: 66.7–68.3 °C. 1H NMR
(400 MHz, DMSO d6): d 6.36 (d, J = 12.0 Hz, 1H), 6.28 (t, J = 8.0 Hz,
1H), 4.12 (q, J = 12.0 Hz, 1H), 3.80 (dd, J = 8.0 Hz, J = 4.0 Hz, 2H),
3.62 (s, 3H), 3.07 (t, J = 4.0 Hz, 1H), 1.76–1.66 (m, 1H), 1.38–1.29
(m, 1H), 1.17–1.06 (m, 1H), 0.86–0.82 (m, 6H); ESI-MS m/z 227.4
[M+H]+.
5.1.1.2. Methyl (S)-2-phenyl-2-(3-(prop-2-yn-1-yl)ureido)acetate
(5c). White solid, yield: 75%, mp: 112.4–114.2 °C. 1H NMR (400
MHz, DMSO d6): d 7.41–7.33 (m, 5H), 6.95 (d, J = 8 Hz, 1H), 6.36
(t, J = 8 Hz, 1H), 5.28 (d, J = 8 Hz, 1H), 3.81 (dd, J = 8 Hz, 4 Hz, 2H),
3.62 (s, 3H), 3.08 (t, J = 4 Hz, 1H); ESI-MS m/z 247.4 [M+H]+.
5.1.1. (S)-Methyl 4-methyl-2-(3-(prop-2-yn-1-yl)ureido)pentanoate
(5a)
Compound 4a (1.81 g, 10 mmol) was dissolved in a mixture
solution of DCM (40 mL) and saturated NaHCO3 (40 mL) followed
by the addition of triphosgene (0.98 g, 3.3 mmol) gradually. The
mixture was stirred in an ice-bath for 1.5 h. The organic layer
was separated and dried over MgSO4 for 15 min. After filtration,
the residue was dropwise added into the solution of propargy-
lamine (0.55 g, 10 mmol) and Et3N (1.21 g, 12 mmol) in anhydrous
DCM (100 mL) cooled in an ice-bath. After stirring at 25 °C for 12 h,
5.1.2. (S)-N-hydroxy-4-methyl-2-(3-(prop-2-yn-1-yl)ureido)
pentanamide (6a)
The preparation of fresh methanol solution of potassium
hydroxylamine.
Potassium hydroxide (28.00 g, 509 mmol) in anhydrous metha-
nol (70 mL) was dropwise added into the anhydrous methanol
(120 mL) solution of hydroxylamine hydrochloride (23.35 g,
343 mmol) cooled in an ice-bath. The mixture was stirred at 0 °C
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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Table 1
The structures and IC50 values of target compounds against APN from porcine kidney.
Compd
R
IC50
(l
M)a
Compd
R
IC50 (l
M)a
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
H
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-NO2
2.1 0.2
0.67 0.09
3.1 0.2
6.9 0.5
0.88 0.06
3.5 0.3
7.8 0.5
0.76 0.09
2.2 0.1
13u
13v
4-OCH2CH3
2-OCH2Ph
3-OCH2Ph
2,6-diCl
2,6-diCH3
2,6-diOCH3
3,4-diCl
3,4-diOCH3
3,4,5-triOCH3
–
1.5 0.1
0.089 0.007
0.24 0.02
0.58 0.06
0.39 0.03
0.47 0.03
5.5 0.3
2.6 0.2
1.4 0.1
13.6 2.1
11.3 1.7
0.68 0.07
3.7 0.3
13w
13aa
13bb
13cc
13dd
13ee
13ff
14
15
20a
20b
25a
3.6 0.2
0.65 0.08
0.91 0.09
1.9 0.1
–
2-NH2
3-NH2
3.4 0.1
2.2 0.1
13o
13p
13q
3-NO2
4-NO2
3-CN
1.7 0.3
1.0 0.1
7.8 0.4
25b
25c
25d
3.6 0.3
2.2 0.1
1.4 0.2
13r
13s
4-CN
1.4 0.2
25e
25f
3.9 0.3
1.9 0.1
2-OCH2CH3
0.44 0.05
13t
3-OCH2CH3
0.82 0.05
9.4 0.5
25g
25h
0.55 0.04
2.3 0.2
Bestatin
–
a
Assays were performed in triplicate; data are shown as mean SD.
Table 2
The IC50 values of selected compounds against APN on ES-2 and PLC/PRF/5 cell surfaces.
Compd
IC50
(
l
M)a
Compd
IC50 (l
M)a
ES-2
PLC/PRF/5
ES-2
PLC/PRF/5
13h
13k
13s
13v
6.9 0.4
6.5 0.5
2.1 0.1
0.74 0.08
20.6 1.0
15.3 1.2
5.0 0.3
1.3 0.1
13w
25g
Bestatin
0.87 0.1
4.8 0.3
35.5 2.3
4.9 0.4
12.3 1.1
54.7 3.5
a
Assays were performed in triplicate; data are shown as mean SD.
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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 3
The IC50 values of selected compounds against proliferation of some tumor cell lines.
Compd
IC50
(l
M)a
U937
HCT116
A549
ES-2
PLC
K562
MDA-MB-231
PC-3
13v
13w
Bestatin
68.4 6.3
70.3 1.5
>500
81.2 5.6
130.1 12.8
>500
90.0 2.9
128.0 1.0
>500
104.6 11.3
107.9 12.5
>500
92.8 5.6
112.2 4.1
>500
85.0 5.2
99.8 13.1
>500
72.5 3.6
72.6 3.4
>500
119.1 1.8
122.9 7.9
>500
a
Assays were performed in triplicate; data are shown as mean SD.
Fig. 2. (A) Representative images of selected inhibitor 13v on the formation of HUVECs capillary tube-like structure. (B) The inhibition rates of 13v on the tubular structures
formation of HUVECs. Data are expressed as the mean standard deviation from triplicate experiments. *P < 0.05, 13v (100 M) vs bestatin (100 M).
l
l
for 40 min and the precipitate was filtered out to provide a fresh
methanol solution of potassium hydroxylamine.
as a white solid (1.20 g, yield: 53%), mp: 124.5–126.2 °C. 1H NMR
(600 MHz, DMSO d6): d 10.68 (s, 1H), 8.80 (s, 1H), 6.24 (t, J = 5.7
Hz, 1H), 6.16 (d, J = 8.4 Hz, 1H), 4.05 (q, J = 8.4 Hz, 1H), 3.78 (dd,
J = 5.7 Hz, J = 2.4 Hz, 2H), 3.05 (t, J = 2.4 Hz, 1H), 1.54–1.50 (m,
1H), 1.34–1.32 (m, 2H), 0.89–0.84 (m, 6H); ESI-MS m/z 226.3
[MꢂH]ꢂ.
Compound 5a (2.26 g, 10 mmol) was dissolved in the solution
above (20 mL) at 25 °C. The mixture was stirred at 25 °C for 0.5 h
and then the methanol was evaporated. The residue was dissolved
in water and then 10% HCl was added to adjust pH to 6. The mix-
ture was extracted by EtOAc (3 ꢁ 100 mL). The organic layer was
washed with saturated NaHCO3 (3 ꢁ 100 mL) and brine (3 ꢁ 100
mL), and dried over MgSO4. After evaporation of EtOAc under vac-
uum, DCM was added to the residue and then placed into refriger-
ator overnight. The formed precipitation was filtered off to give 6a
5.1.2.1.
(2S,3R)-N-hydroxy-3-methyl-2-(3-(prop-2-yn-1-yl)ureido)
pentanamide (6b). White solid, yield: 51%, mp: 144.6–146.1 °C.
1H NMR (400 MHz, DMSO-d6): d 10.64 (s, 1H), 8.82 (s, 1H), 6.34–
6.28 (m, 1H), 6.22–6.14 (m, 1H), 4.01–3.83 (m, 1H), 3.80–3.77
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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
Fig. 3. (A) Representative images of selected inhibitor 13v on the micro-vessels growth of the rat aortic ring. (B) The inhibition rates of 13v on the micro-vessels growth. Data
are expressed as the mean standard deviation from triplicate experiments. *P < 0.05, 13v (100 M) vs bestatin (100 M).
l
l
(m, 2H), 3.08–3.06 (m, 1H), 1.60–1.25 (m, 2H), 1.07–0.97 (m, 1H),
crude product was used directly in the next reaction without fur-
ther purification.
0.86–0.78 (m, 6H); ESI-MS m/z 228.4 [M+H]+.
Compounds 8b–8u, 8aa–8ff, 10v–10w, 17a–17b and 22a–22h
were prepared using the same procedure described above.
5.1.2.2.
(S)-N-hydroxy-2-phenyl-2-(3-(prop-2-yn-1-yl)ureido)ac-
etamide (6c). White solid, yield: 49%, mp: 136.4–138.2 °C. 1H
NMR (400 MHz, DMSO-d6): d 11.00 (s, 1H), 8.95 (s, 1H), 7.37–
7.30 (m, 5H), 6.96–6.90 (m, 1H), 6.37–6.34 (m, 1H), 5.29 (d, J = 8
Hz, 1H), 3.81 (dd, J = 8 Hz, 4 Hz, 2H), 3.08 (t, J = 4 Hz, 1H); ESI-MS
m/z 248.3 [M+H]+.
5.1.4. 2-(Benzyloxy)benzaldehyde (9v)
2-Hydroxybenzaldehyde 7v (1.22 g, 10 mmol) and benzyl bro-
mide (2.05 g, 12 mmol) were dissolved in DMF (20 mL). After addi-
tion of K2CO3 (2.07 g, 15 mmol), the mixture was heated at 80 °C
for 5 h. The reaction was quenched by addition of ice-cold water
(100 mL). Then the mixture was extracted by EtOAc (3 ꢁ 100
mL). The organic layer was washed by 10% HCl (3 ꢁ 100 mL), water
(3 ꢁ 100 mL) and brine (3 ꢁ 100 mL), and dried over MgSO4 over-
night. EtOAc was evaporated to give compound 9v as colorless
oil (1.80 g, yield: 85%). ESI-MS m/z 213.4 [M+H]+. The crude pro-
duct was used directly in the next reaction without further
purification.
5.1.3. Phenylmethanol (8a)
To a solution of compound 7a (1.06 g, 10 mmol) in methanol
(10 mL) was added NaBH4 (0.57 g, 15 mmol). After stirring for 1 h
while cooled with an ice-water bath, methanol was evaporated
and the residue was dissolved in EtOAc (100 mL). The organic layer
was washed with water (3 ꢁ 100 mL) and brine (3 ꢁ 100 mL), and
dried over MgSO4 overnight. EtOAc was evaporated to give 8a as
colorless oil (2.01 g, yield: 94%). ESI-MS m/z 109.4 [M+H]+. The
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8
J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 5. The FlexX docking result of 13v (A) and 13a (B) in the active site of APN
(PDB: 2DQM). The carbon atoms in white, nitrogen atoms in blue, oxygen atoms in
0
red, hydrogen atoms in light blue and the green ball represents zinc ion. S1, S1 and
S2 represent three hydrophobic pockets, respectively.
0
Fig. 4. Antitumor effect of 13v on hepatoma cell H22 pulmonary metastasis model.
(A) Picture of pulmonary lobes with metastasis nodes. (B) The number of metastatic
pulmonary nodes. Data are expressed as mean SD. *P < 0.05 versus control group.
Compounds 12b–12w, 12aa–12ff and 24a–24h were prepared
using the same procedure described above.
Compound 9w was prepared using the same procedure
described above.
5.1.5. Benzyl methanesulfonate (11a)
5.1.7. (2-Aminophenyl)methanol (18a)
To a solution of compound 8a (1.08 g, 10 mmol) and Et3N (1.52
g, 15 mmol) in anhydrous DCM (100 mL) was added dropwise
methanesulfonyl chloride (1.36 g, 12 mmol) in anhydrous DCM
(20 mL). The mixture was stirred for 12 h while being cooled with
an ice-water bath. DCM was evaporated under vacuum. The resi-
due was dissolved in EtOAc (100 mL) and washed with 10% HCl
(3 ꢁ 100 mL), saturated NaHCO3 (3 ꢁ 100 mL) and brine (3 ꢁ 100
mL), and then dried over MgSO4 overnight. EtOAc was evaporated
to give 11a as yellow oil (1.77 g, yield: 95%). ESI-MS m/z 187.4 [M
+H]+. The crude product was used directly in the next reaction
without further purification.
Compound 17a (1.53 g, 10 mmol) was added into the solution
of Na2Sꢀ9H2O (21.60 g, 90 mmol) in water (100 mL). The mixture
was heated at 100 °C for 3 h. After extraction by EtOAc, the organic
layer was washed by water (3 ꢁ 100 mL) and brine (3 ꢁ 100 mL),
and dried over MgSO4 overnight. EtOAc was evaporated to give
compound 18a as a yellow solid (1.07 g, yield: 87%). ESI-MS m/z
124.5 [M+H]+. The crude product was used directly in the next step
without further purification.
Compound 18b was prepared using the same procedure
described above.
Compounds 11b–11w, 11aa–11ff and 23a–23h were prepared
using the same procedure described above.
5.1.8. 2-(Azidomethyl)aniline (19a)
Compound 18a (1.23 g, 10 mmol), triphenylphosphine (2.62 g,
10 mmol) and NaN3 (0.78 g, 12 mmol) in DMF/CCl4 (16 mL/4 mL)
was heated at 100 °C for 12 h. The mixture was poured into ice-
cold water and extracted by Et2O. The organic layer was washed
with water (3 ꢁ 100 mL) and brine (3 ꢁ 100 mL), and dried over
MgSO4 overnight. Et2O was concentrated to give the crude product.
The product was purified by column chromatography (EtOAc/PE)
to give 19a as yellow oil (0.68 g, yield: 46%). ESI-MS m/z 149.5
[M+H]+.
5.1.6. (Azidomethyl)benzene (12a)
Compound 11a (1.86 g, 10 mmol) was dissolved in DMF (10 mL)
followed by the addition of NaN3 (0.72 g, 11 mmol). The mixture
was stirred at 25 °C for 12 h and then was quenched by being
poured into ice-cold water. The mixture was extracted with EtOAc
(3 ꢁ 100 mL). The organic layer was washed with water (3 ꢁ 100
mL) and brine (3 ꢁ 100 mL), and dried over MgSO4 overnight.
EtOAc was evaporated under vacuum to give 12a as colorless oil
(1.18g, yield: 89%). ESI-MS m/z 134.4 [M+H]+. The crude product
was used directly in the next reaction without further purification.
Compound 19b was prepared using the same procedure
described above.
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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
5.1.9. (S)-2-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methyl pentanamide (13a)
24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for C17H24BrN6O3 [M
+H]+ 439.1093, found 439.1093.
Compounds 6a (0.45 g, 2 mmol) and 12a (0.29 g, 2.2 mmol)
were dissolved in DMSO (16 mL). Sodium ascorbate (59.4 mg, 0.3
mmol) in H2O (2 mL) and CuSO4ꢀ5H2O (25 mg, 0.1 mmol) in H2O
(2 mL) were added into the above solution. The mixture was stirred
at 25 °C for 2 h and poured into water (100 mL) and extracted by
EtOAc (3 ꢁ 100 mL). The organic layer was washed by brine (3 ꢁ
100 mL) and dried over MgSO4 overnight. EtOAc was evaporated
to give colorless oil. The residue was dissolved in DCM and stirred
for 2 h at 25 °C. The precipitate formed was filtered off to give 0.35
g of 13a as a white solid. Yield: 48%, mp: 184.8–186.2 °C. 1H NMR
(600 MHz, DMSO d6): d 10.67 (s, 1H), 8.79 (s, 1H), 7.90 (s, 1H), 7.37
(t, J = 6.6 Hz, 2H), 7.32 (t, J = 6.6 Hz, 1H), 7.30 (d, J = 6.6 Hz, 2H),
6.36 (t, J = 6.0 Hz, 1H), 6.11 (d, J = 8.4 Hz, 1H), 5.56 (s, 2H), 4.21–
4.20 (m, 2H), 4.06 (q, J = 8.4 Hz, 1H), 1.51–1.49 (m, 1H), 1.33–
1.30 (m, 2H), 0.86–0.82 (m, 6H); 13C NMR (100 MHz, DMSO d6): d
169.9, 157.6, 146.6, 136.5, 129.1, 128.5, 128.3, 122.9, 53.1, 49.5,
42.9, 35.4, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for
5.1.9.5. (S)-2-(3-((1-(3-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13f). White solid, yield:
49%, mp: 168.9–170.4 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.79 (s, 1H), 7.95 (s, 1H), 7.55–7.53 (m, 1H), 7.40–7.28
(m, 3H), 6.38 (t, J = 5.7 Hz, 1H), 6.12 (d, J = 8.7 Hz, 1H), 5.58 (s,
2H), 4.22 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 1.55–1.44 (m,
1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.2, 139.2, 129.1, 128.6, 128.4, 128.4,
123.0, 67.0, 49.5, 42.8, 35.4, 24.6, 23.2, 22.5; HRMS (AP-ESI) m/z
calcd for C17H24BrN6O3 [M+H]+ 439.1093, found 439.1090.
5.1.9.6. (S)-2-(3-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13g). White solid, yield:
46%, mp: 178.8–180.3 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.79 (s, 1H), 7.91 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.26 (d,
J = 8.4 Hz, 2H), 6.37 (t, J = 5.7 Hz, 1H), 6.12 (d, J = 9.0 Hz, 1H), 5.55
(s, 2H), 4.21 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 1.57–1.44 (m,
1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.7, 136.0, 132.1, 130.6, 123.0, 121.8,
52.4, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd
for C17H24BrN6O3 [M+H]+ 439.1093, found 439.1091.
C
17H25N6O3 [M+H]+ 361.1988, found 361.1992.
5.1.9.1. (S)-2-(3-((1-(2-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy -4-methylpentanamide (13b). White solid, yield:
52%, mp: 154.6–156.3 °C. 1H NMR (300 MHz, DMSO d6): d 10.66 (s,
1H), 8.79 (s, 1H), 7.87 (s, 1H), 7.54–7.50 (m, 1H), 7.43–7.29 (m, 2H),
7.23–7.19 (m, 1H), 6.38 (t, J = 5.7 Hz, 1H), 6.12 (d, J = 9.0 Hz, 1H),
5.67 (s, 2H), 4.22 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 1.55–1.44
(m, 1H), 1.34–1.30 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100
MHz, DMSO d6): d 169.8, 157.6, 146.5, 133.8, 133.0, 130.9, 130.6
130.0, 128.1, 123.2, 50.9, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6; HRMS
(AP-ESI) m/z calcd for C17H24ClN6O3 [M+H]+ 395.1598, found
395.1590.
5.1.9.7. (S)-N-hydroxy-4-methyl-2-(3-((1-(2-methylbenzyl)-1H-1,2,3-
triazol-4-yl) methyl)ureido)pentanamide (13h). White solid, yield:
48%, mp: 166.6–168.3 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.78 (s, 1H), 7.78 (s, 1H), 7.27–7.16 (m, 3H), 7.09–7.06
(m, 1H), 6.36 (t, J = 5.7 Hz, 1H), 6.11 (d, J = 8.7 Hz, 1H), 5.57 (s,
2H), 4.21 (d, J = 5.7 Hz, 2H), 4.09–4.01 (m, 1H), 2.30 (s, 3H), 1.57–
1.42 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR
(100 MHz, DMSO d6): d 169.8, 157.6, 146.5, 136.7, 134.6, 130.8,
129.1, 128.7, 126.6, 122.9, 51.3, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6,
19.0; HRMS (AP-ESI) m/z calcd for C18H27N6O3 [M+H]+ 375.2145,
found 375.2150.
5.1.9.2. (S)-2-(3-((1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13c). White solid, yield:
55%, mp: 186.8–188.5 °C. 1H NMR (300 MHz, DMSO d6): d 10.67
(s, 1H), 8.80 (s, 1H), 7.95 (s, 1H), 7.41–7.40 (m, 3H), 7.28–7.24
(m, 1H), 6.39 (t, J = 5.4 Hz, 1H), 6.13 (d, J = 8.7 Hz, 1H), 5.59 (s,
2H), 4.22 (d, J = 5.4 Hz, 2H), 4.11–4.03 (m, 1H), 1.55–1.44 (m,
1H), 1.35–1.30 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.7, 138.9, 133.7, 131.1, 128.5, 128.2,
127.1, 123.1, 52.3, 49.6, 42.9, 35.4, 24.6, 23.2, 22.6; HRMS (AP-
ESI) m/z calcd for C17H24ClN6O3 [M+H]+ 395.1598, found 395.1601.
5.1.9.8. (S)-N-hydroxy-4-methyl-2-(3-((1-(3-methylbenzyl)-1H-1,2,3-
triazol-4-yl) methyl)ureido)pentanamide (13i). White solid, yield:
55%, mp: 184.8–186.3 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.79 (s, 1H), 7.88 (s, 1H), 7.28–7.23 (m, 1H), 7.15–7.08
(m, 3H), 6.36 (t, J = 5.7 Hz, 1H), 6.11 (d, J = 8.7 Hz, 1H), 5.51 (s,
2H), 4.21 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 2.28 (s, 3H), 1.57–
1.44 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR
(100 MHz, DMSO d6): d 169.8, 157.6, 146.5, 138.4, 136.4, 129.2,
129.1, 129.0, 125.5, 122.8, 53.1, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6,
21.3; HRMS (AP-ESI) m/z calcd for C18H27N6O3 [M+H]+ 375.2145,
found 375.2151.
5.1.9.3. (S)-2-(3-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13d). White solid, yield:
51%, mp: 180.8–182.5 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.78 (s, 1H), 7.91 (s, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.32 (d,
J = 8.4 Hz, 2H), 6.36 (t, J = 5.4 Hz, 1H), 6.11 (d, J = 8.7 Hz, 1H), 5.57
(s, 2H), 4.28 (d, J = 5.4 Hz, 2H), 4.09–4.01 (m, 1H), 1.57–1.44 (m,
1H), 1.34–1.30 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.7, 135.5, 133.2, 130.3, 129.1, 123.0,
52.3, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd
for C17H24ClN6O3 [M+H]+ 395.1598, found 395.1607.
5.1.9.9. (S)-N-hydroxy-4-methyl-2-(3-((1-(4-methylbenzyl)-1H-1,2,3-
triazol-4-yl) methyl)ureido)pentanamide (13j). White solid, yield:
57%, mp: 180.4–182.2 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.79 (s, 1H), 7.85 (s, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.17 (d,
J = 8.1 Hz, 2H), 6.35 (t, J = 5.7 Hz, 1H), 6.10 (d, J = 9.0 Hz, 1H), 5.50
(s, 2H), 4.20 (d, J = 5.7 Hz, 2H), 4.09–4.01 (m, 1H), 2.28 (s, 3H),
1.57–1.44 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H); 13C
NMR (100 MHz, DMSO d6): d 169.8, 157.6, 146.5, 137.9, 133.5,
129.7, 128.4, 122.7, 53.0, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6, 21.1;
HRMS (AP-ESI) m/z calcd for C18H27N6O3 [M+H]+ 375.2145, found
375.2137.
5.1.9.4. (S)-2-(3-((1-(2-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13e). White solid, yield:
47%, mp: 144.8–146.2 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.78 (s, 1H), 7.87 (s, 1H), 7.69 (dd, J = 7.8 Hz, J = 1.2 Hz,
1H), 7.41 (td, J = 7.8 Hz, J = 1.2 Hz, 1H), 7.31 (td, J = 7.8 Hz, J = 1.8
Hz, 1H), 7.14 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 6.38 (t, J = 5.7 Hz, 1H),
6.12 (d, J = 9.0 Hz, 1H), 5.65 (s, 2H), 4.23 (d, J = 5.7 Hz, 2H), 4.10–
4.02 (m, 1H), 1.57–1.44 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82
(m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 157.6, 146.5,
135.4, 133.3, 130.8, 130.8, 128.7, 123.3, 53.2, 49.5, 42.9, 35.4,
5.1.9.10. (S)-N-hydroxy-2-(3-((1-(2-methoxybenzyl)-1H-1,2,3-tria-
zol-4-yl)methyl) ureido)-4-methylpentanamide (13k). White solid,
yield: 53%, mp: 160.8–162.3 °C. 1H NMR (300 MHz, DMSO d6): d
10.67 (s, 1H), 8.79 (s, 1H), 7.75 (s, 1H), 7.37–7.32 (m, 1H), 7.11–
7.04 (m, 2H), 6.96–6.91 (m, 1H), 6.36 (t, J = 5.7 Hz, 1H), 6.11 (d, J
Please cite this article in press as: Cao J., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.041

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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
= 8.7 Hz, 1H), 5.49 (s, 2H), 4.20 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m,
1H), 3.82 (s, 3H), 1.57–1.44 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82
(m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 157.6, 157.2,
146.2, 130.4, 130.0, 124.0, 122.9, 120.9, 111.7, 56.0, 49.5, 48.6,
42.9, 35.4, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for
5.1.9.16. (S)-2-(3-((1-(3-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N- hydroxy-4-methylpentanamide (13q). White solid, yield:
52%, mp: 180.7–182.3 °C. 1H NMR (300 MHz, DMSO d6): d 10.68 (s,
1H), 8.83 (s, 1H), 7.99 (s, 1H), 7.87–7.81 (m, 2H), 7.65–7.57 (m, 2H),
6.39 (t, J = 5.4 Hz, 1H), 6.13 (d, J = 9.0 Hz, 1H), 5.65 (s, 2H), 4.22 (d, J
= 5.4 Hz, 2H), 4.10–4.02 (m, 1H), 1.55–1.44 (m, 1H), 1.35–1.30 (m,
2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8,
157.6, 146.7, 138.1, 133.4, 132.4, 132.0, 130.5, 123.2, 118.9,
112.1, 52.2, 49.5, 42.8, 35.3, 24.6, 23.2, 22.5; HRMS (AP-ESI) m/z
calcd for C18H24N7O3 [M+H]+ 386.1941, found 386.1937.
C
18H27N6O4 [M+H]+ 391.2094, found 391.2091.
5.1.9.11. (S)-N-hydroxy-2-(3-((1-(3-methoxybenzyl)-1H-1,2,3-tria-
zol-4-yl)methyl) ureido)-4-methylpentanamide (13l). White solid,
yield: 51%, mp: 174.5–176.2 °C. 1H NMR (300 MHz, DMSO d6): d
10.67 (s, 1H), 8.79 (s, 1H), 7.90 (s, 1H), 7.31–7.25 (m, 1H), 6.91–
6.83 (m, 3H), 6.37 (t, J = 5.7 Hz, 1H), 6.11 (d, J = 9.0 Hz, 1H), 5.52
(s, 2H), 4.21 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 3.74 (s, 3H),
1.57–1.44 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H); 13C
NMR (100 MHz, DMSO d6): d 169.8, 159.8, 157.6, 146.5, 137.9,
130.3, 123.0, 120.4, 114.2, 113.8, 55.5, 53.0, 49.5, 42.8, 35.3, 24.6,
23.1, 22.5; HRMS (AP-ESI) m/z calcd for C18H27N6O4 [M+H]+
391.2094, found 391.2084.
5.1.9.17. (S)-2-(3-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13r). White solid, yield:
48%, mp: 172.3–174.1 °C.1H NMR (300 MHz, DMSO d6): d 10.68
(s, 1H), 8.81 (s, 1H), 7.97 (s, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.44 (d,
J = 8.1 Hz, 2H), 6.39 (t, J = 5.7 Hz, 1H), 6.13 (d, J = 9.0 Hz, 1H), 5.70
(s, 2H), 4.28 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 1.57–1.44 (m,
1H), 1.34–1.30 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.7, 142.0, 133.1, 129.1, 123.4, 119.0,
111.3, 52.5, 49.5, 42.8, 35.3, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z
calcd for C18H24N7O3 [M+H]+ 386.1941, found 386.1944.
5.1.9.12. (S)-N-hydroxy-2-(3-((1-(4-methoxybenzyl)-1H-1,2,3-tria-
zol-4-yl)methyl) ureido)-4-methylpentanamide (13m). White solid,
yield: 54%, mp: 164.8–166.3 °C. 1H NMR (300 MHz, DMSO d6): d
10.66 (s, 1H), 8.79 (s, 1H), 7.83 (s, 1H), 7.28 (d, J = 8.7 Hz, 2H),
6.92 (d, J = 8.7 Hz, 2H), 6.35 (t, J = 5.7 Hz, 1H), 6.11 (d, J = 8.7 Hz,
1H), 5.47 (s, 2H), 4.19 (d, J = 5.7 Hz, 2H), 4.09–4.01 (m, 1H), 3.73
(s, 3H), 1.55–1.44 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82 (m, 6H);
13C NMR (100 MHz, DMSO d6): d 169.8, 159.5, 157.5, 146.5,
130.0, 128.4, 122.5, 114.5, 55.6, 52.7, 49.5, 42.9, 35.4, 24.6, 23.2,
22.6; HRMS (AP-ESI) m/z calcd for C18H27N6O4 [M+H]+ 391.2094,
found 391.2097.
5.1.9.18. (S)-2-(3-((1-(2-Ethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13s). White solid, yield:
58%, mp: 154.5–156.1 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.77 (s, 1H), 7.74 (s, 1H), 7.32 (td, J = 7.5 Hz, J = 1.5 Hz,
1H), 7.13 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H),
6.92 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 6.36 (t, J = 5.4 Hz, 1H), 6.11 (d,
J = 8.7 Hz, 1H), 5.48 (s, 2H), 4.20 (d, J = 5.4 Hz, 2H), 4.09–4.02 (m,
3H), 1.55–1.42 (m, 1H), 1.35–1.29 (m, 5H), 0.87–0.82 (m, 6H);
13C NMR (100 MHz, DMSO d6): d 169.8, 157.6, 156.6, 146.2,
130.3, 130.2, 124.2, 123.0, 120.8, 112.5, 64.0, 49.5, 48.8, 42.9,
35.4, 24.6, 23.2, 22.6, 15.0; HRMS (AP-ESI) m/z calcd for
5.1.9.13. (S)-N-hydroxy-4-methyl-2-(3-((1-(2-nitrobenzyl)-1H-1,2,3-
triazol-4-yl) methyl)ureido)pentanamide (13n). Yellow solid, yield:
58%, mp: 168.7–170.5 °C. 1H NMR (300 MHz, DMSO d6): d 10.64
(s, 1H), 8.80 (s, 1H), 8.14 (dd, J = 7.5 Hz, J = 1.2 Hz, 1H), 7.93 (s,
1H), 7.74 (td, J = 7.5 Hz, J = 1.2 Hz, 1H), 7.63 (td, J = 7.5 Hz, J = 1.2
Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.41 (t, J = 5.7 Hz, 1H), 6.15 (d, J
= 8.7 Hz, 1H), 5.94 (s, 2H), 4.25 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m,
1H), 1.55–1.44 (m, 1H), 1.34–1.30 (m, 2H), 0.87–0.82 (m, 6H);
13C NMR (100 MHz, DMSO d6): d 169.8, 157.6, 148.0, 146.7,
134.7, 131.4, 130.4, 130.0, 125.4, 123.7, 50.2, 49.5, 42.9, 35.4,
24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for C17H24N7O5 [M+H]+
406.1839, found 406.1845.
C
19H29N6O4 [M+H]+ 405.2250, found 405.2258.
5.1.9.19. (S)-2-(3-((1-(3-Ethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (13t). White solid, yield:
55%, mp: 156.8–158.3 °C. 1H NMR (400 MHz, DMSO d6): d 10.66
(s, 1H), 8.78 (s, 1H), 7.89 (s, 1H), 7.26 (t, J = 8.0 Hz, J = 1.5 Hz, 1H),
6.89–6.82 (m, 3H), 6.37 (t, J = 8.0 Hz, 1H), 6.11 (d, J = 8.0 Hz, 1H),
5.51 (s, 2H), 4.21 (d, J = 8.0 Hz, 2H), 4.09–3.97 (m, 3H), 1.54–1.46
(m, 1H), 1.34–1.29 (m, 5H), 0.87–0.82 (m, 6H); 13C NMR (100
MHz, DMSO d6): d 169.8, 159.1, 157.6, 146.5, 137.9, 130.3, 122.9,
120.3, 114.7, 114.2, 63.4, 53.1, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6,
15.0; HRMS (AP-ESI) m/z calcd for C19H29N6O4 [M+H]+ 405.2250,
found 405.2258.
5.1.9.14. (S)-N-hydroxy-4-methyl-2-(3-((1-(3-nitrobenzyl)-1H-1,2,3-
triazol-4-yl) methyl)ureido)pentanamide (13o). Yellow solid, yield:
57%, mp: 186.8–188.3 °C. 1H NMR (300 MHz, DMSO d6): d 10.68
(s, 1H), 8.80 (s, 1H), 8.23–8.19 (m, 2H), 8.02 (s, 1H), 7.76 (d, J =
7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 6.40 (t, J = 5.7 Hz, 1H), 6.13 (d,
J = 8.7 Hz, 1H), 5.75 (s, 2H), 4.23 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m,
1H), 1.57–1.44 (m, 1H), 1.34–1.30 (m, 2H), 0.87–0.82 (m, 6H);
13C NMR (100 MHz, DMSO d6): d 169.8, 157.6, 148.3, 146.8,
138.7, 135.1, 130.8, 123.5, 123.3, 123.2, 52.1, 49.5, 42.8, 35.3,
24.6, 23.1, 22.6; HRMS (AP-ESI) m/z calcd for C17H24N7O5 [M+H]+
406.1839, found 406.1845.
5.1.9.20. (S)-2-(3-((1-(4-Ethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N- hydroxy-4- methylpentanamide (13u). White solid,
yield: 51%, mp: 164.6–166.2 °C. 1H NMR (400 MHz, DMSO d6): d
10.65 (s, 1H), 8.78 (s, 1H), 7.83 (s, 1H), 7.26 (d, J = 8.0 Hz, 2H),
6.90 (d, J = 8.0 Hz, 2H), 6.35 (t, J = 8.0 Hz, 1H), 6.09 (d, J = 8.0 Hz,
1H), 5.46 (s, 2H), 4.20 (d, J = 8.0 Hz, 2H), 4.08–3.97 (m, 3H), 1.53–
1.47 (m, 1H), 1.33–1.28 (m, 5H), 0.86–0.82 (m, 6H); 13C NMR
(100 MHz, DMSO d6): d 169.8, 158.8, 157.6, 146.5, 130.0, 128.3,
122.5, 115.0, 63.5, 52.7, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6, 15.0;
HRMS (AP-ESI) m/z calcd for C19H29N6O4 [M+H]+ 405.2250, found
405.2268.
5.1.9.15. (S)-N-hydroxy-4-methyl-2-(3-((1-(4-nitrobenzyl)-1H-1,2,3-
triazol-4-yl) methyl)ureido)pentanamide (13p). Yellow solid, yield:
58%, mp: 176.9–178.4 °C.1H NMR (300 MHz, DMSO d6): d 10.68
(s, 1H), 8.80 (s, 1H), 8.24 (d, J = 8.7 Hz, 2H), 8.00 (s, 1H), 7.52 (d,
J = 8.7 Hz, 2H), 6.40 (t, J = 5.1 Hz, 1H), 6.14 (d, J = 8.7 Hz, 1H), 5.76
(s, 2H), 4.24 (d, J = 5.1 Hz, 2H), 4.10–4.02 (m, 1H), 1.55–1.44 (m,
1H), 1.35–1.30 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 147.6, 146.8, 144.0, 129.4, 124.3, 123.4,
52.2, 49.5, 42.8, 35.3, 24.6, 23.1, 22.6; HRMS (AP-ESI) m/z calcd
for C17H24N7O5 [M+H]+ 406.1839, found 406.1836.
5.1.9.21.
(S)-2-(3-((1-(2-(Benzyloxy)benzyl)-1H-1,2,3-triazol-4-yl)
methyl)ureido)-N- hydroxy-4-methylpentanamide (13v). White
solid, yield: 47%, mp: 124.8–126.5 °C. 1H NMR (300 MHz,
DMSO d6): d 10.66 (s, 1H), 8.78 (s, 1H), 7.72 (s, 1H), 7.48–7.29
(m, 6H), 7.13 (d, J = 7.8 Hz, 2H), 6.94 (t, J = 7.8 Hz, 1H), 6.36
(t, J = 5.7 Hz, 1H), 6.12 (d, J = 9.0 Hz, 1H), 5.55 (s, 2H), 5.17
(s, 2H), 4.21 (d, J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 1.57–1.44
Please cite this article in press as: Cao J., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.041

J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
11
(m, 1H), 1.34–1.29 (m, 2H), 0.86–0.81 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.5, 156.2, 146.1, 137.2, 130.3, 130.2, 128.9,
128.2, 127.9, 124.4, 122.9, 121.1, 112.9, 69.8, 49.5, 48.7, 42.9,
35.3, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for C24H31N6O4
[M+H]+ 467.2407, found 467.2403.
(AP-ESI) m/z calcd for C17H23Cl2N6O3 [M+H]+ 429.1209, found
429.1215.
5.1.9.27. (S)-2-(3-((1-(3,4-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)
methyl)ureido)-N-hydroxy-4-methylpentanamide
(13ee). White
solid, yield: 53%, mp: 160.8–162.5 °C. 1H NMR (300 MHz,
DMSO d6): d 10.67 (s, 1H), 8.80 (s, 1H), 7.86 (s, 1H), 7.00 (s, 1H),
6.93 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.40 (t, J = 5.4 Hz,
1H), 6.15 (d, J = 9.0 Hz, 1H), 5.45 (s, 2H), 4.19 (d, J = 5.4 Hz, 2H),
4.09–4.01 (m, 1H), 3.73 (s, 6H), 1.54–1.43 (m, 1H), 1.34–1.29 (m,
2H), 0.86–0.81 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8,
157.6, 149.2, 149.1, 146.5, 128.7, 122.6, 121.1, 112.5, 112.3, 56.0,
55.9, 53.0, 49.6, 42.8, 35.4, 24.6, 23.2, 22.5; HRMS (AP-ESI) m/z
calcd for C19H29N6O5 [M+H]+ 421.2199, found 421.2203.
5.1.9.22.
(S)-2-(3-((1-(3-(Benzyloxy)benzyl)-1H-1,2,3-triazol-4-yl)
(13w). White
methyl)ureido)-N-hydroxy-4-methylpentanamide
solid, yield: 43%, mp: 154.7–156.1 °C. 1H NMR (300 MHz,
DMSO d6): d 10.66 (s, 1H), 8.78 (s, 1H), 7.89 (s, 1H), 7.45–7.25
(m, 6H), 6.98–6.96 (m, 2H), 6.86 (d, J = 7.5 Hz, 1H), 6.37 (t, J = 5.7
Hz, 1H), 6.11 (d, J = 8.7 Hz, 1H), 5.21 (s, 2H), 5.08 (s, 2H), 4.22 (d,
J = 5.7 Hz, 2H), 4.10–4.02 (m, 1H), 1.55–1.44 (m, 1H), 1.34–1.30
(m, 2H), 0.86–0.82 (m, 6H); 13C NMR (100 MHz, DMSO d6): d
169.8, 158.9, 157.6, 146.5, 138.0, 137.3, 130.3, 128.9, 128.3,
128.2, 122.9, 120.7, 115.1, 114.6, 69.7, 53.0, 49.5, 42.9, 35.4, 24.6,
23.2, 22.6; HRMS (AP-ESI) m/z calcd for C24H31N6O4 [M+H]+
467.2407, found 467.2417.
5.1.9.28. (S)-N-hydroxy-4-methyl-2-(3-((1-(3,4,5-trimethoxybenzyl)-
1H-1,2,3-triazol-4-yl)methyl)ureido)pentanamide
(13ff). White
solid, yield: 56%, mp: 152.9–154.2 °C. 1H NMR (300 MHz,
DMSO d6): d 10.67 (s, 1H), 8.80 (s, 1H), 7.90 (s, 1H), 6.69 (s, 2H),
6.37 (t, J = 5.4 Hz, 1H), 6.12 (d, J = 9.0 Hz, 1H), 5.46 (s, 2H), 4.21
(d, J = 5.4 Hz, 2H), 4.09–4.01 (m, 1H), 3.75 (s, 6H), 3.63 (s, 3H),
1.57–1.43 (m, 1H), 1.34–1.29 (m, 2H), 0.86–0.81 (m, 6H); 13C
NMR (100 MHz, DMSO d6): d 169.8, 161.1, 157.5, 153.4, 146.5,
137.7, 131.8, 122.8, 106.5, 106.1, 60.4, 56.3, 55.6, 53.4, 49.5, 42.9,
35.3, 24.6, 23.1, 22.5; HRMS (AP-ESI) m/z calcd for C20H31N6O6
[M+H]+ 451.2305, found 451.2311.
5.1.9.23.
(S)-2-(3-((1-(2,6-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)
methyl)ureido)-N -hydroxy-4-methylpentanamide (13aa). White
solid, yield: 49%, mp: 184.8–186.4 °C. 1H NMR (300 MHz,
DMSO d6): d 10.66 (s, 1H), 8.78 (s, 1H), 7.80 (s, 1H), 7.58 (d, J =
8.1 Hz, 2H), 7.47 (t, J = 8.1 Hz, 1H), 6.35 (t, J = 5.4 Hz, 1H), 6.11 (d,
J = 9.0 Hz, 1H), 5.76 (s, 2H), 4.20 (d, J = 5.4 Hz, 2H), 4.10–4.02 (m,
1H), 1.57–1.44 (m, 1H), 1.38–1.29 (m, 2H), 0.87–0.82 (m, 6H);
13C NMR (100 MHz, DMSO d6): d 169.8, 157.5, 146.2, 136.3,
132.1, 130.8, 129.4, 122.9, 49.5, 48.9, 42.9, 35.3, 24.6, 23.2, 22.5;
HRMS (AP-ESI) m/z calcd for C17H23Cl2N6O3 [M+H]+ 429.1209,
found 429.1200.
5.1.9.29.
(2S,3R)-2-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy- 3-methylpentanamide (14). White solid, yield:
58%, mp: 176.9–178.6 °C. 1H NMR (600 MHz, DMSO d6): d 10.60
(s, 1H), 8.78 (s, 1H), 7.90 (s, 1H), 7.40–7.29 (m, 5H), 6.47–6.40
(m, 1H), 6.16–6.08 (m, 1H), 5.56 (s, 2H), 4.28–4.15 (m, 2H), 4.03–
3.83 (m, 1H), 1.61–1.23 (m, 2H), 1.07–0.92 (m, 1H), 0.85–0.83
(m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.2, 157.9, 146.5,
136.5, 129.1, 128.5, 128.3, 122.9, 55.2, 54.5, 53.1, 38.2, 35.4, 26.1,
24.9; HRMS (AP-ESI) m/z calcd for C17H25N6O3 [M+H]+ 361.1988,
found 361.2005.
5.1.9.24.
(S)-2-(3-((1-(2,6-Dimethylbenzyl)-1H-1,2,3-triazol-4-yl)
(13bb). White
methyl)ureido)-N-hydroxy-4-methylpentanamide
solid, yield: 55%, mp: 154.6–156.2 °C. 1H NMR (300 MHz,
DMSO d6): d 10.65 (s, 1H), 8.78 (s, 1H), 7.60 (s, 1H), 7.17 (t, J =
7.5 Hz, 1H), 7.08 (d, J = 7.5 Hz, 2H), 6.32 (t, J = 5.4 Hz, 1H), 6.09
(d, J = 9.0 Hz, 1H), 5.56 (s, 2H), 4.18 (d, J = 5.4 Hz, 2H), 4.09–4.04
(m, 1H), 2.33 (s, 6H), 1.56–1.43 (m, 1H), 1.33–1.28 (m, 2H), 0.87–
0.81 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 157.5,
146.1, 138.2, 132.1, 129.0, 128.9, 122.4, 49.4, 48.0, 42.9, 35.3,
24.6, 23.2, 22.5, 19.9; HRMS (AP-ESI) m/z calcd for C19H29N6O3
[M+H]+ 389.2301, found 389.2308.
5.1.9.30. (S)-2-(3-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N- hydroxy-2-phenylacetamide (15). White solid, yield:
65%, mp: 202.4–204.2 °C. 1H NMR (400 MHz, DMSO d6): d 11.00
(s, 1H), 9.00 (s, 1H), 7.92 (s, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.37–
7.30 (m, 6H), 7.28–7.23 (m, 1H), 6.89 (d, J = 8.7 Hz, 1H), 6.65 (t, J
= 5.7 Hz, 1H), 5.56 (s, 2H), 5.19 (d, J = 8.7 Hz, 1H), 4.28–4.17 (m,
2H); 13C NMR (100 MHz, DMSO d6): d 167.7, 157.3, 146.6, 140.6,
135.5, 133.3, 130.3, 129.2, 128.7, 127.8, 126.9, 123.0, 54.7, 52.4,
5.1.9.25. (S)-2-(3-((1-(2,6-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)
methyl)ureido)-N-hydroxy-4-methylpentanamide
(13cc). White
solid, yield: 52%, mp: 178.7–180.3 °C. 1H NMR (300 MHz,
DMSO d6): d 10.63 (s, 1H), 8.78 (s, 1H), 7.52 (s, 1H), 7.35 (t, J =
8.4 Hz, 1H), 6.72 (d, J = 8.4 Hz, 2H), 6.32 (t, J = 5.4 Hz, 1H), 6.09
(d, J = 9.0 Hz, 1H), 5.44 (s, 2H), 4.16 (d, J = 5.4 Hz, 2H), 4.09–4.01
(m, 1H), 3.81 (s, 6H), 1.54–1.43 (m, 1H), 1.33–1.29 (m, 2H), 0.87–
0.81 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 158.8,
157.5, 146.0, 131.2, 122.1, 110.9, 104.6, 56.4, 49.5, 42.8, 42.3,
35.3, 24.6, 23.2, 22.5; HRMS (AP-ESI) m/z calcd for C19H29N6O5
[M+H]+ 421.2199, found 421.2207.
35.4; HRMS (AP-ESI) m/z calcd for
C
19H2°ClN6O3 [M+H]+
415.1285, found 415.1282.
5.1.9.31. (S)-2-(3-((1-(2-Aminobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (20a). White solid, yield:
30%, mp: 142.6–144.3 °C. 1H NMR (600 MHz, DMSO d6): d 10.66
(s, 1H), 8.78 (s, 1H), 7.79 (s, 1H), 7.03 (t, J = 7.8 Hz, 1H), 6.94 (d, J
= 7.8 Hz, 1H), 6.67 (d, J = 7.8 Hz, 1H), 6.53 (t, J = 7.8 Hz, 1H), 6.36
(t, J = 5.4 Hz, 1H), 6.10 (d, J = 9 Hz, 1H), 5.42 (s, 2H), 5.23 (s, 2H),
4.21–4.20 (m, 2H), 4.07–4.04 (m, 1H), 1.51–1.48 (m, 1H), 1.32–
1.30 (m, 2H), 0.86–0.83 (m, 6H); 13C NMR (100 MHz, DMSO d6): d
169.8, 157.5, 147.0, 146.3, 130.3, 129.6, 122.8, 119.3, 116.7,
115.8, 50.0, 49.5, 42.9, 35.3, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z
calcd for C17H26N7O3 [M+H]+ 376.2097, found 376.2102.
5.1.9.26.
(S)-2-(3-((1-(3,4-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)
(13dd). White
methyl)ureido)-N-hydroxy-4-methylpentanamide
solid, yield: 48%, mp: 178.7–180.3 °C. 1H NMR (400 MHz,
DMSO d6): d 10.68 (s, 1H), 8.78 (s, 1H), 7.88 (s, 1H), 7.70 (d, J =
4.0 Hz, 1H), 7.47 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 7.25 (d, J = 8.0 Hz,
1H), 6.37 (t, J = 8.0 Hz, 1H), 6.12 (d, J = 8.0 Hz, 1H), 5.66 (s, 2H),
4.22 (d, J = 8.0 Hz, 2H), 4.10–4.03 (m, 1H), 1.54–1.45 (m, 1H),
1.34–1.30 (m, 2H), 0.87–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.5, 134.4, 134.1, 132.9, 132.3, 129.6,
128.3, 123.3, 50.3, 49.5, 42.9, 42.3, 35.3, 24.6, 23.2, 22.6; HRMS
5.1.9.32. (S)-2-(3-((1-(3-Aminobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (20b). White solid, yield:
23%, mp: 88.9–90.2 °C. 1H NMR (600 MHz, DMSO d6): d 10.66 (s,
1H), 8.79 (s, 1H), 7.81 (s, 1H), 6.98 (t, J = 7.8 Hz, 1H), 6.48 (d, J =
7.8 Hz, 1H), 6.44 (s, 1H), 6.41 (d, J = 7.8 Hz, 1H), 6.34 (t, J = 5.4 Hz,
Please cite this article in press as: Cao J., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.041

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J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
1H), 6.11 (d, J = 9.0 Hz, 1H), 5.36 (s, 2H), 5.15 (s, 2H), 4.21–4.17 (m,
2H), 4.07–4.03 (m, 1H), 1.52–1.48 (m, 1H), 1.33–1.30 (m, 2H),
0.86–0.83 (m, 6H); 13C NMR (100 MHz, DMSO-d6): d 169.8, 157.6,
149.4, 146.4, 136.9, 129.6, 122.8, 115.5, 114.0, 113.4, 53.5, 49.6,
42.8, 35.4, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for
(AP-ESI) m/z calcd for C
21H27N6O3 [M+H]+ 411.2145, found
411.2154.
5.1.9.38.
(2S)-N-hydroxy-4-methyl-2-(3-((1-(1-phenylethyl)-1H-
1,2,3-triazol-4-yl) methyl)ureido)pentanamide (25f). White solid,
yield: 49%, mp: 110.7–112.6 °C. 1H NMR (400 MHz, DMSO d6): d
10.56 (s, 1H), 8.79 (s, 1H), 7.96 (s, 1H), 7.39–7.30 (m, 5H), 6.35 (t,
J = 8.0 Hz, 1H), 6.10 (d, J = 8.0 Hz, 1H), 5.91 (q, J = 8.0 Hz, 1H),
4.21 (d, J = 8.0 Hz, 2H), 4.09–4.03 (m, 1H), 1.86 (d, J = 8.0 Hz, 3H),
1.56–1.48 (m, 1H), 1.34–1.30 (m, 2H), 0.88–0.83 (m, 6H); 13C
NMR (100 MHz, DMSO d6): d 169.8, 157.6, 146.2, 141.4, 129.1,
128.4, 126.8, 121.6, 59.5, 49.5, 42.8, 35.4, 24.6, 23.2, 22.6, 21.4;
HRMS (AP-ESI) m/z calcd for C18H27N6O3 [M+H]+ 375.2145, found
375.2156.
C
17H27N7O3 [M+H]+ 376.2097, found 376.2091.
5.1.9.33.
(S,E)-2-(3-((1-Cinnamyl-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy-4-methylpentanamide (25a). White solid, yield:
45%, mp: 178.9–180.5 °C. 1H NMR (300 MHz, DMSO d6): d 10.66
(s, 1H), 8.79 (s, 1H), 7.88 (s, 1H), 7.47–7.45 (m, 2H), 7.37–7.25
(m, 3H), 6.66 (d, J = 15.9 Hz, 1H), 6.48 (d, J = 15.9 Hz, 1H), 6.39 (t,
J = 5.4 Hz, 1H), 6.13 (d, J = 8.7 Hz, 1H), 5.14 (d, J = 6.3 Hz, 2H),
4.23 (d, J = 5.4 Hz, 2H), 4.10–4.02 (m, 1H), 1.57–1.42 (m, 1H),
1.34–1.30 (m, 2H), 0.86–0.82 (m, 6H); 13C NMR (100 MHz,
DMSO d6): d 169.8, 157.6, 146.5, 136.2, 134.1, 129.1, 128.5, 127.0,
124.1, 122.7, 51.7, 49.5, 42.9, 35.4, 24.6, 23.2, 22.6; HRMS (AP-
ESI) m/z calcd for C19H27N6O3 [M+H]+ 387.2145, found 387.2146.
5.1.9.39. (2S)-N-hydroxy-2-(3-((1-(1-(2-methoxyphenyl)ethyl)-1H-
1,2,3-triazol-4-yl)methyl)ureido)-4-methylpentanamide
(25g).
White solid, yield: 41%, mp: 142.5–144.1 °C. 1H NMR (300 MHz,
DMSO d6): d 10.65 (s, 1H), 8.78 (s, 1H), 7.86 (s, 1H), 7.31 (t, J =
8.7 Hz, 1H), 7.06 (t, J = 8.7 Hz, 2H), 6.94 (t, J = 8.7 Hz, 1H), 6.34 (t,
J = 5.4 Hz, 1H), 6.12 (q, J = 6.9 Hz, 1H), 6.10 (d, J = 9.0 Hz, 1H),
4.21 (d, J = 5.4 Hz, 2H), 4.10–4.02 (m, 1H), 3.81 (s, 3H), 1.80 (d, J
= 6.9 Hz, 3H), 1.55–1.47 (m, 1H), 1.34–1.29 (m, 2H), 0.87–0.82
(m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 157.6, 156.3,
145.8, 129.8, 129.0, 126.9, 121.9, 121.0, 111.8, 56.1, 53.9, 49.5,
42.8, 35.4, 24.6, 23.2, 22.5, 20.7; HRMS (AP-ESI) m/z calcd for
5.1.9.34. (S)-N-hydroxy-4-methyl-2-(3-((1-phenethyl-1H-1,2,3-tria-
zol-4-yl) methyl)ureido)pentanamide (25b). White solid, yield:
41%, mp: 178.4–180.1 °C. 1H NMR (300 MHz, DMSO d6): d 10.69
(s, 1H), 8.82 (s, 1H), 7.83 (s, 1H), 7.31–7.19 (m, 5H), 6.36 (t, J =
5.4 Hz, 1H), 6.14 (d, J = 9.0 Hz, 1H), 4.57 (d, J = 7.5 Hz, 2H), 4.20
(d, J = 5.4 Hz, 2H), 4.11–4.03 (m, 1H), 3.13 (d, J = 7.5 Hz, 2H),
1.57–1.46 (m, 1H), 1.36–1.31 (m, 2H), 0.89–0.84 (m, 6H); 13C
NMR (100 MHz, DMSO d6): d 169.8, 157.6, 146.0, 138.1, 129.1,
128.8, 127.0, 122.8, 50.7, 49.6, 42.9, 36.2, 35.3, 24.6, 23.2, 22.6;
HRMS (AP-ESI) m/z calcd for C18H27N6O3 [M+H]+ 375.2145, found
375.2159.
C
19H29N6O4 [M+H]+ 405.2250, found 405.2257.
5.1.9.40. (S)-2-(3-((1-Benzhydryl-1H-1,2,3-triazol-4-yl)methyl)
ureido)-N-hydroxy -4-methylpentanamide (25h). White solid, yield:
40%, mp: 178.4–180.3 °C. 1H NMR (400 MHz, DMSO d6): d 10.67 (s,
1H), 8.79 (s, 1H), 7.83 (s, 1H), 7.42–7.33 (m, 6H), 7.28 (s, 1H), 7.21–
7.19 (m, 4H), 6.38 (t, J = 8.0 Hz, 1H), 6.11 (d, J = 8.0 Hz, 1H), 4.24 (d,
J = 8.0 Hz, 2H), 4.09–4.03 (m, 1H), 1.55–1.43 (m, 1H), 1.33–1.30 (m,
2H), 0.87–0.81 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8,
157.5, 146.7, 146.6, 139.2, 136.5, 131.4, 131.4, 131.1, 129.2,
128.5, 128.4, 127.5, 123.1, 122.9, 122.2, 52.3, 49.5, 42.9, 35.3,
24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for C23H29N6O3 [M+H]+
437.2301, found 437.2300.
5.1.9.35. (S)-N-hydroxy-4-methyl-2-(3-((1-(pyridin-3-ylmethyl)-1H-
1,2,3-triazol-4-yl)methyl)ureido)pentanamide (25c). White solid,
yield: 49%, mp: 144.6–146.3 °C. 1H NMR (600 MHz, DMSO d6): d
10.66 (s, 1H), 8.80 (s, 1H), 8.58 (s, 1H), 8.55–8.54 (m, 1H), 7.97
(s, 1H), 7.72–7.70 (m, 1H), 7.41–7.39 (m, 1H), 6.37 (t, J = 5.4 Hz,
1H), 6.11 (d, J = 9.0 Hz, 1H), 5.62 (s, 2H), 4.21 (d, J = 5.4 Hz, 2H),
4.07–4.03 (m, 1H), 1.51–1.48 (m, 1H), 1.33–1.30 (m, 2H), 0.86–
0.83 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 157.6,
149.8, 149.6, 146.7, 136.2, 132.2, 124.2, 123.0, 50.6, 49.5, 42.9,
35.4, 24.6, 23.2, 22.6; HRMS (AP-ESI) m/z calcd for C16H24N7O3
[M+H]+ 362.1941, found 362.1940.
5.2. Biological evaluation
5.2.1. APN inhibition assay
5.1.9.36. (S)-N-hydroxy-4-methyl-2-(3-((1-(naphthalen-1-ylmethyl)-
IC50 values against APN were determined by using L-Leu-p-
nitroanilide as substrate and microsomal APN from porcine kidney
microsomes (Biocol) in 50 mM PBS, pH 7.2 as enzyme. The inhibi-
1H-1,2,3-triazol-4-yl)methyl)ureido)pentanamide
(25d). White
solid, yield: 46%, mp: 178.5–180.2 °C. 1H NMR (300 MHz,
DMSO d6): d 10.66 (s, 1H), 8.79 (s, 1H), 8.21–8.18 (m, 1H), 8.00–
7.94 (m, 2H), 7.83 (s, 1H), 7.62–7.48 (m, 3H), 7.44–7.42 (m, 1H),
6.34 (t, J = 5.4 Hz, 1H), 6.09 (d, J = 9.0 Hz, 1H), 6.06 (s, 2H), 4.18
(d, J = 5.4 Hz, 2H), 4.07–4.00 (m, 1H), 1.52–1.43 (m, 1H), 1.32–
1.27 (m, 2H), 0.85–0.80 (m, 6H); 13C NMR (100 MHz, DMSO d6): d
169.8, 157.5, 146.5, 133.8, 132.0, 131.1, 129.4, 129.1, 127.7,
127.2, 126.6, 126.0, 123.7, 123.0, 51.1, 49.5, 42.8, 35.3, 24.6, 23.2,
22.5; HRMS (AP-ESI) m/z calcd for C21H27N6O3 [M+H]+ 411.2145,
found 411.2150.
tors (40
(10 L) at 37 °C for 5 min. Then, substrate (5
the above solution and PBS was added to adjust the assay mixture
to 200 L. The mixture was incubated at 37 °C for another 30 min.
l
L) at various concentrations were incubated with APN
l
lL) was added to
l
The hydrolysis product p-nitroanilide was measured according to
the absorbance monitored at 405 nm with a plate reader (Var-
ioskan, Thermo, USA).
5.2.2. Enzyme inhibition assay towards APN from ES-2 or PLC/PRF/5
cells
5.1.9.37. (S)-N-hydroxy-4-methyl-2-(3-((1-(naphthalen-2-ylmethyl)-
1H-1,2,3-triazol-4-yl)methyl)ureido)pentanamide
(25e). White
IC50 values against APN were determined by using L-Leu-p-
solid, yield: 51%, mp: 174.8–176.4 °C. 1H NMR (600 MHz,
DMSO d6): d 10.65 (s, 1H), 8.78 (s, 1H), 7.94–7.90 (m, 4H), 7.86
(s, 1H), 7.57–7.51 (m, 2H), 7.45–7.41 (m, 1H), 6.36 (t, J = 5.4 Hz,
1H), 6.10 (d, J = 8.7 Hz, 1H), 5.73 (s, 2H), 4.22 (d, J = 5.4 Hz, 2H),
4.08–4.00 (m, 1H), 1.53–1.44 (m, 1H), 1.33–1.28 (m, 2H), 0.84–
0.80 (m, 6H); 13C NMR (100 MHz, DMSO d6): d 169.8, 157.5,
146.6, 134.0, 133.2, 132.9, 128.9, 128.2, 128.0, 127.4, 127.0,
126.8, 126.2, 123.0, 53.3, 49.5, 42.8, 35.4, 24.6, 23.1, 22.5; HRMS
nitroanilide as substrate and APN on the surface of ES-2 cells or
PLC/PRF/5 cells in 10 mM PBS, pH 7.4 as enzyme. The inhibitors
(20
l
L) were incubated with ES-2 cells or PLC/PRF/5 cells (70
l
L,
2.8 ꢁ 106/mL) at 37 °C for 5 min. Then, substrate (10
lL) and 1 ꢁ
PBS buffer (100 lL) was added to the above solution. The mixture
was incubated at 37 °C for 1 h. After centrifugation, the hydrolysis
product p-nitroanilide in the supernant was monitored at 405 nm
with a plate reader (Varioskan, Thermo, USA).
Please cite this article in press as: Cao J., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.041

J. Cao et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
13
5.2.3. Anti-proliferation assay
formed to further optimize the protein structure. The molecular
structures were generated with Sybyl/Sketch module and opti-
mized using Powell’s method with the Tripos force field with con-
vergence criterion set at 0.05 kcal/Å mol and assigned with the
Gasteiger-Hückel method. Molecular docking was carried out via
the Sybyl/Surflex-Dock module. Other docking parameters implied
in the program were default values. The top-scoring pose was
selected for discussions.
Anti-proliferation abilities of compounds were evaluated by the
MTT [(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
bromide)] method. Cells were grown in RPMI 1640 medium with
10% FBS at 37 °C in 5% CO2 humidified incubator. In brief, cells
(100
then treated with different concentrations of inhibitors (100
for 48 h. MTT (20 L, 5 mg/mL) solution was added into the test
well. After incubation for another 4 h, DMSO (200 L) was added
lL) were plated in 96-well plates and cultivated for 4 h, and
l
L)
l
l
and mixed for 15 min to extract the formed formazan from MTT.
The optical density was then measured using a plate reader at
570 nm (Varioskan, Thermo, USA).
5.4. Statistical analysis
The statistical significance of differences between the groups
was assessed by Student’s t test. P < 0.05 was considered as statis-
tically significant.
5.2.4. HUVEC tubular structure formation assay
Matrigel (50
well plates and then allowed to polymerize for 0.5 h at 37 °C.
HUVECs (20,000 cells/well) in M199 medium (50 L) and inhibi-
tors (50 L) at the specified concentrations were added and incu-
bated at 37 °C in 5% CO2 for 4 h. Images of five random fields per
well were analyzed by Motic Image Plus 2.0 software (Motic
Instruments Inc., Canada). The numbers of branch points of the
formed tubes were counted and the average numbers were calcu-
lated. Experiments were repeated three times.
lL; BD biosciences) was added into test well of 96-
Acknowledgements
l
l
This work was supported by Natural Science Foundation of
Shandong Province (Grant No. ZR2018QH007), National Natural
Science Foundation of China (Grant No. 81373282), Major Project
of Science and Technology of Shandong Province (Grant No.
2015ZDJS04001, 2017CXGC1401), Young Scholars Program of
Shandong University (YSPSDU, Grant No. 2016WLJH33).
5.2.5. Rat aortic ring assay
Conflict of interest
Matrigel (100 lL; BD bioscience) was added into 96-well plates
and then allowed to polymerizefor 0.5 h at 37 °C. The thoracic aortas
were separated from 8- to 10-week-old male Sprague Dawley rats.
After careful removalof fibroadiposetissues, the aortas were cut into
1-mm-long cross-section. The aortas fragments were placed into
test well of 96-well plates and then covered with an additional
The authors confirm that this article content has no conflict of
interest.
A. Supplementary data
100 lL of matrigel. After incubation at 37 °C and 5% CO2 for 0.5 h,
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.04.041.
the matrigel solidified. The aortas rings were treated with inhibitors
at 37 °C in 5% CO2 for 9 days. The micro-vessels formed and were
visualized by inverted microscope at 100ꢁ magnification. The cul-
ture medium containing inhibitors was changed every three days.
The quantity of assay is valued by relative area covered with
microvessels using Image-pro Plus 6.0.27 Experiments were
repeated three times using aortas from four different rats.
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All experiments involving laboratory animals were performed
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Please cite this article in press as: Cao J., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.04.041