Leucine ureido derivatives as aminopeptidase N inhibitors using click chemistry. Part II

Article abstract of DOI:10.1016/j.bmc.2019.01.041

Aminopeptidase N (APN) has been proved to be deeply associated with cancer angiogenesis, metastasis and invasion. Therefore, APN gains increasing attention as a promising anti-tumor target. In the current study, we report the design, synthesis, biological evaluation and structure-activity relationship of one new series of leucine ureido derivatives containing the 1,2,3-triazole moiety. Among them, compound 31f was identified as the best APN inhibitor with IC₅₀ value being two orders of magnitude lower than that of the positive control bestatin. Compound 31f possessed selective cytotoxicity to several tumor cell lines over the normal cell line human umbilical vein endothelial cells (HUVECs). Notably, when combined with 5-fluorouracil (5-Fu), 31f exhibited synergistic anti-proliferation effect against several tumor cell lines. At the same concentration, 31f exhibited much better anti-angiogenesis activities than bestatin in the HUVECs capillary tube formation assay and the rat thoracic aorta rings test. In the in vitro anti-invasion assay, 31f also exhibited superior potency over bestatin. Moreover, considerable in vivo antitumor potencies of 31f alone or in combination with 5-Fu were observed without significant toxic signs in a mouse heptoma H22 tumor transplant model.

Full text of DOI:10.1016/j.bmc.2019.01.041

Accepted Manuscript
Leucine Ureido Derivatives as Aminopeptidase N Inhibitors Using Click Chem-
istry. Part II
Jiangying Cao, Jie Zang, Xiujie Kong, Chunlong Zhao, Ting Chen, Yingying
Ran, Hang Dong, Wenfang Xu, Yingjie Zhang
PII:
S0968-0896(18)32168-0
https://doi.org/10.1016/j.bmc.2019.01.041
BMC 14733
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 December 2018
27 January 2019
30 January 2019
Please cite this article as: Cao, J., Zang, J., Kong, X., Zhao, C., Chen, T., Ran, Y., Dong, H., Xu, W., Zhang, Y.,
Leucine Ureido Derivatives as Aminopeptidase N Inhibitors Using Click Chemistry. Part II, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.041
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Leucine Ureido Derivatives as Aminopeptidase N Inhibitors Using
Click Chemistry. Part II
Jiangying Caoa, Jie Zanga, Xiujie Konga, Chunlong Zhaoa, Ting Chena,
Yingying Rana, Hang Donga, Wenfang Xua, Yingjie Zhanga,*
aDepartment of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong
University, Jinan 250012, P. R. China
*Corresponding Author. Tel/Fax: +86 531 88382009
E-mail address: zhangyingjie@sdu.edu.cn (Y. Zhang).

Abstract
Aminopeptidase N (APN) has been proved to be deeply associated with cancer
angiogenesis, metastasis and invasion. Therefore, APN gains increasing attention as a
promising anti-tumor target. In the current study, we report the design, synthesis,
biological evaluation and structure-activity relationship of one new series of leucine
ureido derivatives containing the 1,2,3-triazole moiety. Among them, compound 31f
was identified as the best APN inhibitor with IC50 value being two orders of magnitude
lower than that of the positive control bestatin. Compound 31f possessed selective
cytotoxicity to several tumor cell lines over the normal cell line human umbilical vein
endothelial cells (HUVECs). Notably, when combined with 5-fluorouracil (5-Fu), 31f
exhibited synergistic anti-proliferation effect against several tumor cell lines. At the
same concentration, 31f exhibited much better anti-angiogenesis activities than bestatin
in the HUVECs capillary tube formation assay and the rat thoracic aorta rings test. In
the in vitro anti-invasion assay, 31f also exhibited superior potency over bestatin.
Moreover, considerable in vivo antitumor potencies of 31f alone or in combination with
5-Fu were observed without significant toxic signs in a mouse heptoma H22 tumor
transplant model.
Key words: aminopeptidase N; CD13; anti-angiogenesis; anti-metastasis; triazole.

1. Introduction
Aminopeptidase N (APN; CD13; EC 3.4.11.2) is a widely expressed Zn2+ dependent
membrane-bound exopeptidase belonging to the M1 family of the MA clan of
peptidase.1,2 The full length 967 amino acids of aminopeptidase N can be divided into
three parts: a short cytoplasmic domain, a single transmembrane part and a large
extracellular domain containing the active site.3 It is known to preferentially release
basic or neutral amino acids from the N-terminus of the biologically active peptides.4
APN is a moonlighting protein possessing the enzymatic as well as other functions,
such as antigen presentation and the receptor for some viruses.5 Enhanced APN
expression has been observed on diverse malignancies, such as melanoma6, renal7,
colon8, prostate9 and gastric10 carcinomas. The expression of APN is related to the
regulation of signal peptides and is associated with the tumor migration, invasion,
metastasis and angiogenesis.11-17 It was revealed that APN could degrade the
extracellular matrix (ECM) to promote malignant cells invasion and metastasis.15,16
Recently, it was reported that the APN expressed by both cancer cells and nonmalignant
stromal cells within the tumor environment could cooperatively promote the
angiogenesis.17 Moreover, APN is a functional marker of semi-dormant liver cancer
cells that are responsible for chemotherapy resistance and cancer relapse.18
Because of the significant role of APN in cancer, the rational design of potent APN
inhibitors as antitumor agents is of considerable interest. To date, several classes of
APN inhibitors (APNIs) have been reported (Figure 1), including AHPA ((2S, 3R)-3-
amino-2-hydroxy-4-phenylbutanoic acid) derivative 1 (bestatin), hydroxamic acid
derivative 2 (tosedostat), mercaptan derivative 3 (PC18), amino-benzosuberone
derivative 4, boronic acid derivative 5, aminophosphinic derivative 6 (PL253), L-
isoserine derivative 7, flavone derivative 8, dicarbonyl derivative 9, L-arginine

derivative 10, chloramphenicol amine derivative 11 and 3-phenylpropane-1,2-diamine
derivative 12.19-21
Figure 1. Structures of representative APNIs.
So far, bestatin, with an AHPA skeleton, has been approved as the adjuvant for the
treatment of adult acute non-lymphocytic leukaemia. Recently, bestatin was found to
have broad spectrum of biological activities, such as anti-angiogenic, anti-metastatic,
and immunomodulatory effects.22-23 Our group has been pursuing the development of
APNIs as anticancer agents for a long time.24-30 In our previous work, the leucine ureido
derivatives with the 1,2,3-triazole moiety (13, Figure 2) exhibited excellent APN
inhibitory potency and promising in vitro and in vivo anti-angiogenesis and anti-
metastasis effect.31 Herein, to extend the structure-activity relationships (SARs) of these
leucine ureido derivatives, a new series of analogues 14 was designed and synthesized.

Compared with 13, the terminal substituted phenyl group of 14 (Figure 2) was directly
attached to the triazole moiety to investigate the effect of conformational restriction on
APN inhibition. Besides, one carboxylic acid derivative and two isoleucine and
methionine derivatives (Figure 2) were also synthesized to respectively investigate the
effects of the zinc binding group (ZBG) and the side chain on APN inhibition.
Figure 2. The structure of bestatin and the design strategy of novel APNIs.
2. Chemistry
The synthetic routes of target compounds are shown in Schemes 1-4. As shown in
Scheme 1, compounds 15a-15c reacted with triphosgene to generate isocyanates, which
were then immediately reacted with propargylamine to yield the ureido derivatives 16a-
16c. The ureido derivatives 16a-16c reacted with sodium hydroxide or NH2OK to
generate the key intermediates 17 or 18a-18c, respectively.

Scheme 1. Reagents and conditions: (a) triphosgene, NaHCO3, DCM/H2O, 0 °C, 1.5 h; (b)
propargylamine, TEA, DCM, 25 °C, 12 h; (c) 16a for 17, NaOH, H2O, MeOH, 25 °C, 0.5 h; (d)
NH2OK, MeOH, 25 °C, 0.5 h.
As shown in Scheme 2, activation of the amino groups of the aromatic amines 19a-
19z and 19aa with sodium nitrite in 10% hydrochloric acid and then reaction with
sodium azide led to the azide derivatives 20a-20z and 20aa. Compound 20a was
coupled with 17, 18b or 18c via click chemistry to give target compounds 21, 23 or 24,
respectively. The target compounds 22a-22z and 22aa were respectively generated by
coupling 18a with 20a-20z and 20aa via click chemistry.
Scheme 3 presented the synthesis of the key intermediate 28. Compound 27 was
prepared using compound 25 as the starting material according to the method reported
before.31 Activation of the amino group of compound 27 with sodium nitrite in 10%
hydrochloric acid and then reaction with sodium azide led to the key intermediate 28.
Compound 28 reacted with 29a-29i to generate compounds 30a-30i, which were then
coupled with 18a to give the target compounds 31a-31i (Scheme 4).

Scheme 2. Reagents and conditions: (a) i: 10% HCl, NaNO2, 0 °C, 0.5 h, ii: NaN3, 0 °C, 0.5 h; (b)
.
17 for 20a, CuSO4 5H2O, sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h; (c) 18a for 20a-20z or
.
.
20aa, CuSO4 5H2O, sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h; (d) 18b for 20a, CuSO4 5H2O,
.
sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h; (e) 18c for 20a, CuSO4 5H2O, sodium ascorbate,
DMSO/H2O (4:1), 25 °C, 2 h.

Scheme 3. Reagents and conditions: (a) NaBH4, MeOH, 0 °C, 1 h; (b) Na2S.9H2O, H2O, 100 °C, 3
h; (c) i: 10% HCl, NaNO2, 0 °C, 0.5 h, ii: NaN3, 0 °C, 0.5 h.
Scheme 4. Reagents and conditions: (a) 28, Et3N, THF, 50 °C, 3 h; (b) 18a for 30a-30i,
.
CuSO4 5H2O, sodium ascorbate, DMSO/H2O (4:1), 25 °C, 2 h.
3. Results and discussion
3.1. Enzyme inhibitory activities of the target compounds against porcine APN
All the target compounds synthesized were firstly evaluated for their inhibitory
activities against porcine APN (Microsomal, Biocol) with bestatin as the positive
control. The results in Table 1 showed that compound 22a was over 18-fold more potent
than bestatin. It was also revealed that 22a exhibited 4-fold and 7-fold improved APN
inhibitory activities relative to 13a and 13b, respectively, indicating the beneficial effect
of conformational restriction on APN inhibition. The carboxylic acid derivative 21
exhibited much less APN inhibitory activity than its corresponding hydroxamic acid
derivative 22a, suggesting the significant role of hydroxamic acid as the strong ZBG in

APN inhibition. Moreover, comparing 22a with 23 and 24, it was easy to find that the
replacement of leucine residue with isoleucine or methionine residue was detrimental to
APN inhibition. Therefore, 22a was selected as the lead compound for further structural
optimization and modification. Introduction of various substituents on the terminal
phenyl group of 22a led to mono-substituted analogs 22b-22r, and dual-substituted
analogs 22s-22z and 22aa. For compounds with single halogen (22b-22g), single
methyl (22h-22j) or single methoxyl (22n-22p) substituents, the ortho-substituted
compounds (22b, 22e, 22h, 22n) exhibited better activities than their meta- (22c, 22f,
22i, 22o) or para-substituted (22d, 22g, 22j, 22p) analogs. However, this trend was not
observed in compounds with single hydroxyl (22k-22m) substituents. Generally, mono-
substituents on the terminal phenyl group of 22a were tolerated, leading to several
compounds (22b, 22e, 22l, 22m, 22n, 22q, 22r) with improved activities relative to
22a, however, most of the dual-substituents were detrimental, especially compared with
their corresponding mono-substituents. Note that the most potent mono-substituted
analogs (22q, 22r) and the most potent dual-substituted analog (22w) all possessed
larger substituents at the ortho-position. Therefore, analogs with extra-large ortho-
substituents (31a-31i) were synthesized and evaluated. Satisfyingly, all these
compounds except 31g exhibited better APN inhibitory activities than 22a. The most
potent compound 31f (IC50 = 0.032 ± 0.003 μM) was over 15-fold more potent than 22a
(IC50 = 0.51 ± 0.02 μM) and over 293-fold more potent than the positive control bestatin
(IC50 = 9.4 ± 0.5 μM).
Table 1. The structures and IC50 values of target compounds against APN from porcine kidney.

IC50 (M)a
IC50 (M)a
Compd
R
--
Compd
R
2,6-diCH3
2,6-diCl
2,5-diCl
2,6-diOCH3
2,6-diOCH2CH3
3,5-diCl
3,4-diCH3
3-Cl-4-CH3
3,5-diCH3
--
2.1 ± 0.2b
3.6 ± 0.3b
98.9 ± 8.6
0.51 ± 0.02
0.28 ± 0.02
1.9 ± 0.2
1.5 ± 0.3
0.36 ± 0.03
1.1 ± 0.2
13a
13b
21
22s
22t
--
--
22u
22v
22w
22x
22y
22z
22aa
23
H
0.58 ± 0.05
0.30 ± 0.01
3.6 ± 0.2
22a
22b
22c
22d
22e
22f
22g
22h
22i
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CH3
3-CH3
1.2 ± 0.3
1.8 ± 0.1
0.40 ± 0.01
1.9 ± 0.1
2.7 ± 0.2
1.7 ± 0.2
1.7 ± 0.2
19.3 ± 1.4
4.1 ± 0.4
0.60 ± 0.05
4.8 ± 0.4
--
24
CH3
0.42 ± 0.04
31a
4-CH3
2-OH
2.5 ± 0.3
0.12 ± 0.01
0.16 ± 0.02
22j
31b
31c
0.52 ± 0.01
22k
3-OH
4-OH
0.34 ± 0.03
0.30 ± 0.01
0.27 ± 0.02
0.26 ± 0.01
22l
31d
31e
22m

2-OCH3
0.47 ± 0.01
0.032 ± 0.003
22n
31f
3-OCH3
4-OCH3
0.54 ± 0.05
0.50 ± 0.03
1.2 ± 0.2
22o
22p
31g
31h
0.33 ± 0.02
2-COOCH3
2-OCH2CH3
0.25 ± 0.01
0.26 ± 0.03
0.085 ± 0.004
9.4 ± 0.5
22q
22r
31i
Bestatin
--
aAssays were performed in triplicate; data are shown as mean ± SD. bReported in reference 31.
3.2. Enzyme inhibitory activities of the selected compounds against human APN on the
surface of ES-2 and PLC/PRF/5 cells
Furthermore, we evaluated the inhibitory potency of selected compounds 31b, 31c,
31d, 31f and 31i against human APN located on the surface of the ES-2 and PLC/PRF/5
cells. Both cell lines have been proved to have relatively higher APN expression on the
cell membrane. The results are listed in Table 2. All the selected compounds exhibited
much better activities than the positive control bestatin (IC50 = 35.5 ± 2.3 μM for ES-2
cell surface APN, IC50 = 54.7 ± 3.5 μM for PLC/PRF/5 cell surface APN), and the
human APN inhibitory order is 31f > 31i > 31b > 31c > 31d, which was consistent with
the results of inhibition against porcine APN. The most potent compound 31f possessed
IC50 values of 0.39 ± 0.01 μM and 0.81 ± 0.04 μM against APN on the surface of ES-2
and PLC/PRF/5 cells, respectively.
Table 2. The IC50 values of selected compounds against APN on ES-2 and PLC/PRF/5 cell surfaces.
IC50 (μM)a
IC50 (μM)a
Compd
Compd
ES-2
PLC/PRF/5
1.4 ± 0.3
2.3 ± 0.2
3.3 ± 0.3
ES-2
PLC/PRF/5
0.81 ± 0.04
1.2 ± 0.1
0.65 ± 0.07
0.99 ± 0.08
1.1 ± 0.1
0.39 ± 0.01
0.62 ± 0.03
35.5 ± 2.3
31b
31c
31d
31f
31i
Bestatin
54.7 ± 3.5
aAssays were performed in triplicate; data are shown as mean ± SD.

3.3. In vitro anti-proliferative activities of the selected compounds
Compound 31f with the most potent porcine and human APN inhibitory activity was
further evaluated in the anti-proliferation assay against six tumor cell lines (PLC, K562,
A549, ES-2, PC-3 and H7402) and one normal cell line (the human umbilical vein
endothelial cell, HUVEC). The results in Table 3 showed that 31f exhibited much
superior anti-proliferative potencies than bestatin against all the tested tumor cell lines.
Importantly, the IC50 value of 31f against HUVEC was > 2000 μM, demonstrating its
selective cytotoxicity against tumor cells over normal cells.
Recently, bestatin was reported to show synergistic anti-tumor effects in combination
with chemotherapy drugs, such as 5-fluorouracil (5-Fu), cisplatin (CDDP), and
doxorubicin (DXR).32 Encouraged by this, we also evaluated the anti-proliferative
effects of 31f plus 5-Fu. The combination index (CI) calculated based on the
Chou−Talalay method was used to evaluate the combined effects of 31f and 5-Fu.33 As
shown in Table 3, the CI values for all the tested tumor cell lines were less than 1,
indicating the synergistic effects of the combination of 31f and 5-Fu in inhibiting the
proliferation of tumor cells.
Table 3. In vitro antiproliferative activities of selected compounds.
IC50 (μM)a
Compd
PLC
K562
A549
ES-2
PC-3
H7402
HUVEC
64.3 ± 3.1 40.6 ± 0.2 57.5 ± 5.5 61.2 ± 3.5 63.2 ± 1.5 52.6 ± 4.3 > 2000
31f
5-Fu
116.8 ± 5.4 20.7 ± 5.7 38.7 ± 8.4 37.9 ± 4.9 49.1 ± 5.8 76.3 ± 6.8
> 500 > 500 > 500 > 500 > 500 > 500
NDb
> 2000
NDb
Bestatin
31f + 5-Fuc 17.1 ± 1.0 6.9 ± 0.4 16.6 ± 1.7 10.3 ± 1.0 17.6 ± 2.2 15.2 ± 0.4
(CI = 0.4)d (CI = 0.5) (CI = 0.7) (CI = 0.4) (CI = 0.6) (CI = 0.5)
b
c
a Assays were performed in triplicate; data are shown as mean ± SD. Not determined. Combined
at the mole ratio of 1:1. d CI > 1 indicates antagonism, CI = 1 indicates an additive effect, and CI < 1
indicates synergism.

3.4. In vitro and ex vivo anti-angiogenesis assays
To compare the anti-angiogenic effects of compound 31f and bestatin, the in vitro
HUVEC tubular structure formation assay was performed. As shown in Figure 3A, both
bestatin and 31f could obviously decrease the HUVEC tubular structure formation at
their noncytotoxic concentrations (the IC50 values of bestatin and 31f against HUVEC
were over 2000 μM, as shown in Table 3). Moreover, compound 31f inhibited the
HUVEC tube formation in a dose-dependent manner, the inhibiting rate of compound
31f at 10 μM was comparable to that of bestatin at 100 μM (Figure 3B), revealing the
superior anti-angiogenic activity of 31f. When the concentration of 31f was increased to
100 μM, no obvious tubular structures were observed.
Control
Bestatin (100 μM)
A
31f (10 μM)
31f (100 μM)

B
Figure 3. (A) Representative images of the capillary-like tubular network of HUVECs treated with
DMSO (0.5%) or compounds. (B) The inhibition rates of tested compounds on the HUVEC tubular
structure formation. Data are expressed as the mean ± standard deviation from triplicate
experiments. * P < 0.05.
The ex vivo rat thoracic aorta ring assay was used to further evaluate the anti-
angiogenic activity of compound 31f. As shown in Figure 4, 31f exhibited the capacities
of inhibiting the micro-vessels growth in a dose-dependent manner. At the
concentration of 100 μM, 31f almost completely inhibited the micro-vessels growth,
much more potent than bestatin.
Control
Bestatin (100 μM)
A
31f (10 μM)
31f (100 μM)

B
Figure 4. (A) Representative images of the rat thoracic aorta rings treated with DMSO (0.5%) or
compounds. (B) The inhibition rates of tested compounds on the micro-vessels growth. Data are
expressed as the mean ± standard deviation from triplicate experiments. * P < 0.05.
3.5. In vitro anti-invasion assay
Tumor cell invasion plays an important role in the progress of cancer metastasis. In
this research, an anti-invasion assay was performed on transwell chambers coated with
Matrigel. As shown in Figure 5, ES-2 cell could freely invade and pass through
Matrigel in the negative control group, while this progress was dramatically impeded by
31f and bestatin at their noncytotoxic concentrations (the IC50 values of 31f and bestatin
against ES-2 cells were 61.2 ± 3.5 μM and ＞ 500 μM, respectively, as shown in Table
3). Actually, after 8 h treatment (the time needed for the anti-invasion assay) with 10
μM of 31f, the growth inhibition rate of ES-2 cells was around 1.4%, which confirmed
that the anti-invasion effect of 31f was not due to its cytotoxicity. Notably, 10 μM of
31f exhibited comparable, if not better anti-invasion activity than 100 μM of bestatin.

Control
Bestatin (100 μM)
31f (10 μM)
A
B
Figure 5. (A) Representative images of ES-2 cell invasion treated with DMSO (0.5%) or
compounds. (B) The inhibition rates of tested compounds on the ES-2 cell invasion. Data are
expressed as the mean ± standard deviation from triplicate experiments.
3.6. In vivo antitumor evaluation
Encouraged by its promising antiproliferation, anti-angiogenesis and anti-invasion
potencies, 31f was progressed to the in vivo antitumor evaluation in a mouse heptoma
H22 tumor transplant model. As shown in Figure 6A and 6B, intraperitoneal
administration of 31f alone or in combination with 5-Fu could effectively inhibit tumor
growth. It was worth noting that 31f (82 mg/kg, equivalent to 154 μmol/kg) plus 5-Fu
(20 mg/kg, equivalent to 154 μmol/kg) exhibited more potent anti-tumor activity than
31f (82 mg/kg, equivalent to 154 μmol/kg) alone or 5-Fu (20 mg/kg, equivalent to 154
μmol/kg) alone, which was in line with their synergistic antiproliferation activity shown
in Table 3. What’s more, at the same molar concentration, the in vivo anti-tumor
potency of 31f (82 mg/kg, equivalent to 154 μmol/kg) plus 5-Fu (20 mg/kg, equivalent

to 154 μmol/kg) was similar to that of bestatin (47 mg/kg, equivalent to 154 μmol/kg)
plus 5-Fu (20 mg/kg, equivalent to 154 μmol/kg). During the experiment, the body
weights of mice were monitored every two days. Interestingly, in contrast to the mice
group treated with 5-Fu alone, no body weight loss was observed in mice groups treated
with 31f alone or in combination with 5-Fu (Figure 6C).
A
C
B
Figure 6. In vivo antitumor effects in a mouse heptoma H22 tumor transplant model. (A) The
pictures of dissected tumor tissues. (B) Tumor growth inhibition rate. (C) Mean mice body weights
monitored every two days.
4. Conclusion
In summary, extended SARs of previously reported leucine ureido-based APNI were
conducted and led to abundant compounds with better APN inhibitory potency than the
positive control bestatin. In vitro biological evaluation of the most potent compound 31f
revealed its potent anti-proliferation, anti-angiogenesis and anti-invasion activities. The

following in vivo antitumor evaluation showed that 31f alone or in combination with 5-
Fu could effectively inhibit tumor growth in a mouse heptoma H22 tumor transplant
model. These results support the further research and development of 31f as a promising
antitumor lead compound.
5. Experimental section
5.1. Chemistry: General procedures
All the commercially available materials (Adamas-beta) were used without further
purification otherwise noted. All reactions were monitored by thin-layer
chromatography (TLC) on 0.25 mm silica gel plates (60 GF-254) and visualized by UV
light, ferric chloride and iodine vapor. Column chromatography and recrystallization
were used to purify the products. Melting points were determined on an electrothermal
1
melting point apparatus without correction. H-NMR and 13C-NMR spectra were
determined on a Brucker DRX spectrometer with TMS as an internal standard.
Chemical shifts were described as δ in parts per million and J in Hertz. HRMS and ESI-
MS were conducted by Shandong Analysis and Test Center.
5.1.1. Preparation of compounds 16a-16c
Compounds methyl (prop-2-yn-1-ylcarbamoyl)-L-leucinate (16a) and methyl (prop-
2-yn-1-ylcarbamoyl)-L-alloisoleucinate (16b) were obtained as described previously.31
5.1.1.1. (S)-Methyl 4-(methylthio)-2-(3-(prop-2-yn-1-yl)ureido)butanoate (16c)
According to the similar procedures described by our group31, compound 15c was
converted to 16c as a white solid (2.07 g, yield 85%), mp: 70.6-72.2 °C. 1H NMR (400
MHz, DMSO-d6): δ 6.49 (d, J = 8.0 Hz, 1H), 6.30 (t, J = 8.0 Hz, 1H), 4.30-4.25 (m, 1H),
3.79 (dd, J = 8.0 Hz, J = 4.0 Hz, 2H), 3.63 (s, 3H), 3.06 (t, J = 4.0 Hz, 1H), 2.48-2.44
(m, 2H), 2.04 (s, 3H), 1.95-1.76 (m, 2H); ESI-MS m/z 245.4 [M+H]+.
5.1.2. (S)-4-Methyl-2-(3-(prop-2-yn-1-yl)ureido)pentanoic acid (17)

Compound 16a (2.26 g, 10 mmol) was added into the mixture of 1N NaOH aqueous
solution (12 mL) and methanol (12 mL). After stirring at 25 °C for 0.5 h, 10 % HCl was
added to adjust pH to 6. The aqueous layer was extracted by EtOAc and then dried over
MgSO4. After filtration, EtOAc was evaporated and the residue was dissolved in DCM.
The mixture was placed into refrigerator for 12 h. The formed precipitate was filtered
off to give 1.34 g of 17 as a white solid. Yield: 63%, mp: 76.4-78.2 °C. 1H NMR (400
MHz, DMSO-d6): δ 12.50 (s, 1H), 6.28-6.23 (m, 2H), 4.15-4.11 (m, 1H), 3.80-3.78 (m,
2H), 3.06-3.05 (m, 1H), 1.66-1.59 (m, 1H), 1.48-1.39 (m, 2H), 0.89-0.87 (m, 6H); ESI-
MS m/z 213.4 [M+H]+.
5.1.3. Preparation of compounds 18a-18c
Compounds (S)-N-hydroxy-4-methyl-2-(3-(prop-2-yn-1-yl)ureido)pentanamide (18a)
and (2S,3R)-N-hydroxy-3-methyl-2-(3-(prop-2-yn-1-yl)ureido)pentanamide (18b) were
obtained as described previously.31
5.1.3.1. (S)-N-hydroxy-4-(methylthio)-2-(3-(prop-2-yn-1-yl)ureido)butanamide (18c)
According to the similar procedure described by our group31, compound 16c was
1
converted to 18c as a white solid (1.35 g, yield 55%), mp: 88.6-90.2 °C. H NMR (600
MHz, DMSO-d6): δ 10.68 (s, 1H), 8.86 (s, 1H), 6.31-6.29 (m, 2H), 4.11-4.08 (m, 1H),
3.79-3.77 (m, 2H), 3.06-3.05 (m, 1H), 2.42-2.33 (m, 2H), 2.03 (s, 3H), 1.79-1.66 (m,
2H); ESI-MS m/z 246.4 [M+H]+.
5.1.4. Preparation of compounds 20a-20z and 20aa
5.1.4.1. Azidobenzene (20a)
To a solution of aniline (0.93 g, 10 mmol) in 10% HCl (50 mL) cooled in an ice-bath
was added NaNO2 (0.74 g, 10.7 mmol). The mixture was stirred at 0 °C for 0.5 h and
followed by the addition of NaN3 (0.72 g, 11 mmol). The mixture was stirred at 0 °C for
0.5 h and the aqueous layer was extracted by EtOAc (3 × 50 mL). The organic layer

was dried over MgSO4 overnight. After filtration, EtOAc was evaporated to give 20a as
yellow oil (1.09 g, yield 92%), which was used directly in the next reaction without
further purification.
Compounds 20b-20z and 20aa were prepared from compounds 19b-19z and 19aa in
a similar manner as described for compound 20a, respectively.
5.1.5. Preparation of compounds 21, 22a-22z, 22aa, 23 and 24
5.1.5.1.
(S)-4-Methyl-2-(3-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)ureido)pentanoic
acid (21)
To a solution of compounds 17 (0.42 g, 2 mmol) and 20a (0.26 g, 2.2 mmol) in
DMSO (16 mL) was added sodium ascorbate (59.4 mg, 0.3 mmol) in H2O (2 mL) and
.
CuSO4 5H2O (25 mg, 0.1 mmol) in H2O (2 mL). The mixture was stirred at 25 °C for 2
h. After addition of water (100 mL), the mixture was extracted by EtOAc (3 × 100 mL).
The organic layer was washed with brine (3 × 100 mL) and dried over MgSO4. EtOAc
was evaporated and the residue was dissolved in DCM (30 mL). The mixture was
stirred at 25 °C for 2 h. The formed precipitate was filtered off to give 0.36 g of
1
compound 21 as a white solid. Yield: 55%, mp: 140.4-142.2 °C. H NMR (300 MHz,
DMSO-d6): δ 12.48 (s, 1H), 8.57 (s, 1H), 7.89-7.85 (m, 2H), 7.62-7.57 (m, 2H), 7.51-
7.46 (m, 1H), 6.49 (t, J = 5.7 Hz, 1H), 6.26 (d, J = 8.4 Hz, 1H), 4.35-4.32 (m, 2H), 4.17-
13
4.09 (m, 1H), 1.71-1.58 (m, 1H), 1.48-1.40 (m, 2H), 0.90-0.85 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 175.2, 157.5, 146.9, 136.6, 129.8, 128.5, 120.7, 119.9, 50.9, 41.0,
34.8, 24.2, 22.7, 21.5; HRMS (AP-ESI) m/z calcd for C16H22N5O3 [M+H]+ 332.1723,
found 332.1733.
Compounds 22a-22z and 22aa were prepared by coupling compounds 20a-20z and
20aa with 18a in a similar manner as described for compound 21, respectively.
5.1.5.2. (S)-N-hydroxy-4-methyl-2-(3-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)ureido)

pentanamide (22a)
1
White solid, yield: 47%, mp: 174.2-176.2 °C. H NMR (300 MHz, DMSO-d6): δ
10.69 (s, 1H), 8.81 (s, 1H), 8.58 (s, 1H), 7.90-7.87 (m, 2H), 7.62-7.57 (m, 2H), 7.51-
7.46 (m, 1H), 6.48 (t, J = 5.7 Hz, 1H), 6.18 (d, J = 8.7 Hz, 1H), 4.34-4.31 (m, 2H), 4.08
(q, J = 8.7 Hz, 1H), 1.66-1.43 (m, 1H), 1.37-1.32 (m, 2H), 0.88-0.84 (m, 6H); 13C-NMR
(75 MHz, DMSO-d6): δ 169.4, 157.2, 146.9, 136.6, 129.8, 128.5, 120.6, 119.9, 49.1,
42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for C16H23N6O3 [M+H]+
347.1832, found 347.1826.
5.1.5.3.
(S)-2-(3-((1-(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxyl-4-methylpentanamide (22b)
1
White solid, yield: 61%, mp: 144.2-146.0 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.31 (s, 1H), 7.77 (dd, J = 7.8 Hz, J = 1.2 Hz, 1H), 7.66 (dd,
J = 7.8 Hz, J = 1.2 Hz, 1H), 7.63 (td, J = 7.8 Hz, J = 1.2 Hz, 1H),7.58 (td, J = 7.8 Hz, J
= 1.2 Hz, 1H), 6.49 (t, J = 5.4 Hz, 1H), 6.20 (d, J = 9.0 Hz, 1H), 4.36-4.34 (m, 2H),
4.09 (q, J = 9.0 Hz, 1H), 1.56-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); 13C-
NMR (75 MHz, DMSO-d6): δ 169.3, 157.1, 145.9, 134.5, 131.4, 130.5, 128.4, 128.3,
128.2, 124.5, 49.0, 42.3, 34.7, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for
C16H22ClN6O3 [M+H]+ 381.1442, found 381.1435.
5.1.5.4. (S)-2-(3-((1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-hydroxy-
4-methylpentanamide (22c)
1
White solid, yield: 58%, mp: 176.4-178.4 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.68 (s, 1H), 8.03 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.62 (t, J
= 8.4 Hz, 1H),7.56 (d, J = 8.4 Hz, 1H), 6.48 (t, J = 5.4 Hz, 1H), 6.18 (d, J = 9.0 Hz,
1H), 4.34-4.31 (m, 2H), 4.07 (q, J = 9.0 Hz, 1H), 1.55-1.51 (m, 1H), 1.36-1.33 (m, 2H),
0.88-0.84 (m, 6H); 13C-NMR (75 MHz, DMSO-d6): δ 169.4, 157.2, 147.1, 137.6, 134.1,

131.6, 128.3, 120.9, 119.6, 118.4, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI)
m/z calcd for C16H22ClN6O3 [M+H]+ 381.1442, found 381.1453.
5.1.5.5.
(S)-2-(3-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22d)
1
White solid, yield: 62%, mp: 180.2-182.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.69 (s, 1H), 8.81 (s, 1H), 8.62 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz,
2H), 6.48 (t, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 4.34-4.31 (m, 2H), 4.07 (q, J =
13
9.0 Hz, 1H), 1.55-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 169.3, 157.2, 147.1, 135.4, 132.7, 129.8, 121.5, 120.8, 49.1, 42.3,
34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for C16H22ClN6O3 [M+H]+ 381.1442,
found 381.1445.
5.1.5.6. (S)-2-(3-((1-(2-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-hydroxy-
4-methylpentanamide (22e)
1
White solid, yield: 66%, mp: 164.4-166.4 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.79 (s, 1H), 8.28 (s, 1H), 7.92-7.91 (m, 1H), 7.62-7.54 (m, 3H), 6.48 (t, J
= 5.4 Hz, 1H), 6.20 (d, J = 9.0 Hz, 1H), 4.36-4.33 (m, 2H), 4.08 (q, J = 9.0 Hz, 1H),
1.56-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); 13C-NMR (75 MHz, DMSO-
d6): δ 169.3, 157.1, 145.8, 136.2, 133.6, 131.8, 128.9, 128.5, 124.5, 118.6, 49.1, 42.3,
34.7, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for C16H22BrN6O3 [M+H]+ 425.0937,
found 425.0927.
5.1.5.7.
(S)-2-(3-((1-(3-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22f)
1
White solid, yield: 59%, mp: 176.4-178.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.68 (s, 1H), 8.15 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.69 (d,
J = 7.8 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 6.48 (t, J = 5.4 Hz, 1H), 6.18 (d, J = 9.0 Hz,

1H), 4.34-4.31 (m, 2H), 4.07 (q, J = 9.0 Hz, 1H), 1.55-1.51 (m, 1H), 1.36-1.33 (m, 2H),
0.88-0.85 (m, 6H); 13C-NMR (75 MHz, DMSO-d6): δ 169.3, 157.1, 147.1, 137.7, 131.8,
131.2, 122.4, 120.9, 118.8, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z
calcd for C16H22BrN6O3 [M+H]+ 425.0937, found 425.0944.
5.1.5.8.
(S)-2-(3-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22g)
1
White solid, yield: 64%, mp: 188.2-190.0 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.62 (s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz,
2H), 6.48 (t, J = 5.4 Hz, 1H), 6.18 (d, J = 9.0 Hz, 1H), 4.34-4.30 (m, 2H), 4.07 (q, J =
13
9.0 Hz, 1H), 1.54-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 169.3, 157.1, 147.1, 135.8, 132.7, 121.8, 121.1, 120.7, 49.1, 42.3,
34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for C16H22BrN6O3 [M+H]+ 425.0937,
found 425.0928.
5.1.5.9. (S)-N-hydroxy-4-methyl-2-(3-((1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)
pentanamide (22h)
1
White solid, yield: 63%, mp: 130.4-132.4 °C. H NMR (600 MHz, DMSO-d6): δ
10.67 (s, 1H), 8.79 (s, 1H), 8.22 (s, 1H), 7.48-7.47 (m, 2H), 7.41-7.40 (m, 2H), 6.46 (t, J
= 5.4 Hz, 1H), 6.17 (d, J = 9.0 Hz, 1H), 4.34-4.32 (m, 2H), 4.08 (q, J = 9.0 Hz, 1H),
13
2.14 (s, 3H), 1.54-1.52 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 169.3, 157.1, 145.7, 136.3, 132.9, 131.3, 129.6, 126.9, 125.8,
124.0, 49.1, 42.3, 34.8, 24.1, 22.7, 22.0, 17.3; HRMS (AP-ESI) m/z calcd for
C17H25N6O3 [M+H]+ 361.1988, found 361.1985.
5.1.5.10.
ureido)pentanamide (22i)
White solid, yield: 58%, mp: 174.2-176.0 °C. H NMR (600 MHz, DMSO-d6): δ
(S)-N-hydroxy-4-methyl-2-(3-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methyl)
1

10.67 (s, 1H), 8.79 (s, 1H), 8.22 (s, 1H), 7.48-7.47 (m, 2H), 7.41-7.40 (m, 2H), 6.46 (t, J
= 5.4 Hz, 1H), 6.17 (d, J = 9.0 Hz, 1H), 4.34-4.32 (m, 2H), 4.08 (q, J = 9.0 Hz, 1H),
13
2.14 (s, 3H), 1.54-1.52 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 169.3, 157.1, 145.7, 136.3, 132.9, 131.3, 129.6, 126.9, 125.8,
124.0, 49.1, 42.3, 34.8, 24.1, 22.7, 22.0, 17.3; HRMS (AP-ESI) m/z calcd for
C17H25N6O3 [M+H]+ 361.1988, found 361.1985.
5.1.5.11.
(S)-N-hydroxy-4-methyl-2-(3-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)pentanamide (22j)
1
White solid, yield: 66%, mp: 166.4-168.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.67 (s, 1H), 8.79 (s, 1H), 8.51 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz,
2H), 6.46 (t, J = 5.4 Hz, 1H), 6.17 (d, J = 9.0 Hz, 1H), 4.32-4.30 (m, 2H), 4.08 (q, J =
9.0 Hz, 1H), 2.38 (s, 3H), 1.54-1.52 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H);
13C-NMR (75 MHz, DMSO-d6): δ 169.4, 157.2, 146.7, 138.1, 134.4, 130.1, 120.5,
119.7, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1, 20.5; HRMS (AP-ESI) m/z calcd for
C17H25N6O3 [M+H]+ 361.1988, found 361.1986.
5.1.5.12.
(S)-N-hydroxy-2-(3-((1-(2-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-4-methylpentanamide (22k)
White solid, yield: 43%, mp: 80.4-82.4 °C. 1H NMR (600 MHz, DMSO-d6): δ 10.68
(s, 1H), 10.54 (s, 1H), 9.20 (s, 1H), 8.81 (s, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.33 (t, J =
7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.45 (t, J = 5.4 Hz, 1H),
6.17 (d, J = 8.4 Hz, 1H), 4.33-4.31 (m, 2H), 4.07 (q, J = 8.4 Hz, 1H), 1.55-1.50 (m, 1H),
13
1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75 MHz, DMSO-d6): δ 169.3, 157.1,
149.8, 145.5, 129.8, 124.8, 124.5, 123.8, 119.1, 117.1, 49.0, 42.3, 34.8, 24.1, 22.7, 22.1;
HRMS (AP-ESI) m/z calcd for C16H23N6O3 [M+H]+ 363.1781, found 363.1784.
5.1.5.13.
(S)-N-hydroxy-2-(3-((1-(3-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)

ureido)-4-methylpentanamide (22l)
1
White solid, yield: 48%, mp: 164.4-166.4 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 10.04 (s, 1H), 8.80 (s, 1H), 8.50 (s, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.27-
7.26 (m, 2H), 6.86 (d, J = 7.8 Hz, 1H), 6.45 (t, J = 5.4 Hz, 1H), 6.17 (d, J = 8.4 Hz, 1H),
4.32-4.30 (m, 2H), 4.08 (q, J = 8.4 Hz, 1H), 1.55-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-
0.84 (m, 6H); 13C-NMR (75 MHz, DMSO-d6): δ 169.3, 158.4, 157.1, 146.7, 137.6,
130.7, 120.6, 115.4, 110.2, 106.8, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI)
m/z calcd for C16H23N6O3 [M+H]+ 363.1781, found 363.1790.
5.1.5.14.
(S)-N-hydroxy-2-(3-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-4-methylpentanamide (22m)
1
White solid, yield: 49%, mp: 136.4-138.4 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 9.92 (s, 1H), 8.80 (s, 1H), 8.38 (s, 1H), 7.63 (t, J = 8.4 Hz, 1H), 6.92 (t, J
= 8.4 Hz, 2H), 6.86 (d, J = 7.8 Hz, 1H), 6.44 (t, J = 5.4 Hz, 1H), 6.16 (d, J = 9.0 Hz,
1H), 4.31-4.28 (m, 2H), 4.07 (q, J = 9.0 Hz, 1H), 1.55-1.51 (m, 1H), 1.35-1.33 (m, 2H),
0.88-0.84 (m, 6H); 13C-NMR (75 MHz, DMSO-d6): δ 169.4, 157.6, 157.2, 146.4, 128.8,
121.7, 120.5, 115.9, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for
C16H23N6O3 [M+H]+ 363.1781, found 363.1779.
5.1.5.15.
(S)-N-hydroxy-2-(3-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-4-methylpentanamide (22n)
1
White solid, yield: 64%, mp: 130.2-132.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.79 (s, 1H), 8.20 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.8 Hz,
1H), 7.32 (d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.46 (t, J = 5.4 Hz, 1H), 6.19 (d,
J = 9.0 Hz, 1H), 4.33-4.31 (m, 2H), 4.08 (q, J = 9.0 Hz, 1H), 3.85 (s, 3H), 1.56-1.51 (m,
13
1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75 MHz, DMSO-d6): δ 169.3,
157.1, 151.4, 145.6, 130.5, 125.7, 125.5, 124.2, 120.8, 112.9, 56.0, 49.0, 42.3, 34.8,

24.1, 22.7, 22.0; HRMS (AP-ESI) m/z calcd for C17H25N6O4 [M+H]+ 377.1937, found
377.1928.
5.1.5.16.
(S)-N-hydroxy-2-(3-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-4-methylpentanamide (22o)
1
White solid, yield: 68%, mp: 156.2-158.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.67 (s, 1H), 8.79 (s, 1H), 8.61 (s, 1H), 7.50-7.45 (m, 3H), 7.05-7.04 (m, 1H), 6.47 (t, J
= 5.4 Hz, 1H), 6.18 (d, J = 9.0 Hz, 1H), 4.33-4.31 (m, 2H), 4.07 (q, J = 9.0 Hz, 1H),
13
3.86 (s, 3H), 1.55-1.53 (m, 1H), 1.36-1.33 (m, 2H), 0.88-0.85 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 169.4, 160.1, 157.2, 146.8, 137.6, 130.7, 120.8, 114.3, 111.8,
105.4, 55.5, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for
C17H25N6O4 [M+H]+ 377.1937, found 377.1935.
5.1.5.17.
(S)-N-hydroxy-2-(3-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)
ureido)-4-methylpentanamide (22p)
1
White solid, yield: 64%, mp: 178.2-180.0 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.47 (s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz,
2H), 6.45 (t, J = 5.4 Hz, 1H), 6.17 (d, J = 9.0 Hz, 1H), 4.32-4.29 (m, 2H), 4.07 (q, J =
9.0 Hz, 1H), 3.83 (s, 3H), 1.55-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H);
13C-NMR (75 MHz, DMSO-d6): δ 169.4, 159.1, 157.1, 146.6, 130.0, 121.5, 120.6,
114.8, 55.5, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for
C17H25N6O4 [M+H]+ 377.1937, found 377.1928.
5.1.5.18.
(S)-Methyl-2-(4-((3-(1-(hydroxyamino)-4-methyl-1-oxopentan-2-yl)
ureido)methyl)-1H-1,2,3-triazol-1-yl)benzoate (22q)
1
White solid, yield: 66%, mp: 144.4-146.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.30 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.8 Hz,
1H), 7.69 (t, J = 7.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 6.46 (t, J = 5.4 Hz, 1H), 6.21 (d,

J = 9.0 Hz, 1H), 4.34-4.31 (m, 2H), 4.08 (q, J = 9.0 Hz, 1H), 3.62 (s, 3H), 1.55-1.51 (m,
13
1H), 1.35-1.33 (m, 2H), 0.88-0.85 (m, 6H); C-NMR (75 MHz, DMSO-d6): δ 169.3,
165.7, 157.1, 146.0, 135.2, 132.8, 130.3, 129.8, 127.1, 125.9, 123.6, 52.3, 49.0, 42.3,
34.7, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for C18H25N6O5 [M+H]+ 405.1886,
found 405.1889.
5.1.5.19.
(S)-2-(3-((1-(2-Ethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22r)
1
White solid, yield: 70%, mp: 176.4-178.2 °C. H NMR (300 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.79 (s, 1H), 8.22 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz,
1H), 7.30 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.46 (t, J = 5.4 Hz, 1H), 6.18 (d,
J = 8.7 Hz, 1H), 4.33-4.31 (m, 2H), 4.19-4.05 (m, 3H), 1.60-1.47 (m, 1H), 1.37-1.28
(m, 5H), 0.88-0.84 (m, 6H); 13C-NMR (75 MHz, DMSO-d6): δ 169.3, 157.1, 150.3,
145.6, 130.3, 125.8, 125.2, 124.1, 120.8, 113.9, 64.3, 49.0, 42.4, 34.8, 24.1, 22.7, 22.1,
14.3; HRMS (AP-ESI) m/z calcd for C18H27N6O4 [M+H]+ 391.2094, found 391.2089.
5.1.5.20.
(S)-2-(3-((1-(2,6-Dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22s)
1
White solid, yield: 58%, mp: 174.4-176.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.67 (s, 1H), 8.79 (s, 1H), 8.11 (s, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 7.8 Hz,
2H), 6.44 (t, J = 4.8 Hz, 1H), 6.17 (d, J = 9.0 Hz, 1H), 4.34-4.31 (m, 2H), 4.08 (q, J =
9.0 Hz, 1H), 1.91 (s, 6H), 1.55-1.51 (m, 1H), 1.35-1.32 (m, 2H), 0.87-0.84 (m, 6H);
13C-NMR (75 MHz, DMSO-d6): δ 169.3, 157.1, 145.6, 135.8, 134.7, 129.8, 128.3,
124.2, 49.0, 42.4, 34.9, 24.1, 22.7, 22.0, 16.8; HRMS (AP-ESI) m/z calcd for
C18H27N6O3 [M+H]+ 375.2145, found 375.2152.
5.1.5.21.
(S)-2-(3-((1-(2,6-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido
-N-hydroxy-4-methylpentanamide (22t)

1
White solid, yield: 64%, mp: 170.2-172.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.29 (s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 8.4 Hz,
1H), 6.49 (t, J = 5.4 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H), 4.40-4.37 (m, 2H), 4.09 (q, J =
13
8.4 Hz, 1H), 1.55-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); C-NMR (75
MHz, DMSO-d6): δ 169.3, 157.1, 145.9, 132.7, 132.7, 132.5, 129.1, 124.9, 49.0, 42.4,
34.7, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for C16H21Cl2N6O3 [M+H]+
415.1052, found 415.1048.
5.1.5.22.
(S)-2-(3-((1-(2,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22u)
1
White solid, yield: 52%, mp: 164.4-166.4 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.34 (s, 1H), 7.88 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.72 (d,
J = 8.4 Hz, 1H), 6.49 (t, J = 5.4 Hz, 1H), 6.20 (d, J = 9.0 Hz, 1H), 4.36-4.34 (m, 2H),
4.08 (q, J = 9.0 Hz, 1H), 1.55-1.51 (m, 1H), 1.35-1.33 (m, 2H), 0.88-0.84 (m, 6H); 13C-
NMR (75 MHz, DMSO-d6): δ 169.3, 157.1, 146.0, 135.4, 132.4, 131.9, 131.2, 128.0,
127.3, 124.5, 49.0, 42.3, 34.7, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for
C16H21Cl2N6O3 [M+H]+ 415.1052, found 415.1059.
5.1.5.23. (S)-2-(3-((1-(2,6-Dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22v)
1
White solid, yield: 63%, mp: 168.4-170.0 °C. H NMR (300 MHz, DMSO-d6): δ
10.67 (s, 1H), 8.79 (s, 1H), 7.88 (s, 1H), 7.51 (t, J = 8.4 Hz, 1H), 6.87 (d, J = 8.4 Hz,
2H), 6.44 (t, J = 5.4 Hz, 1H), 6.18 (d, J = 9.0 Hz, 1H), 4.32-4.30 (m, 2H), 4.08 (q, J =
9.0 Hz, 1H), 3.72 (s, 6H), 1.61-1.48 (m, 1H), 1.37-1.32 (m, 2H), 0.88-0.84 (m, 6H);
13C-NMR (75 MHz, DMSO-d6): δ 169.4, 157.1, 155.4, 144.8, 131.5, 125.2, 114.6,
104.6, 56.0, 49.1, 42.3, 34.8, 24.1, 22.7, 22.1; HRMS (AP-ESI) m/z calcd for
C18H27N6O5 [M+H]+ 407.2043, found 407.2050.

5.1.5.24.
(S)-2-(3-((1-(2,6-Diethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22w)
1
White solid, yield: 63%, mp: 164.4-166.4 °C. H NMR (300 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.79 (s, 1H), 7.85 (s, 1H), 7.45 (t, J = 8.4 Hz, 1H), 6.84 (d, J = 8.4 Hz,
2H), 6.42 (t, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 4.32-4.30 (m, 2H), 4.13-4.00 (m,
13
5H), 1.61-1.47 (m, 1H), 1.36-1.32 (m, 2H), 1.16-1.11 (s, 6H), 0.88-0.84 (m, 6H); C-
NMR (75 MHz, DMSO-d6): δ 169.3, 157.1, 154.6, 144.5, 131.3, 125.2, 115.5, 105.6,
64.3, 49.0, 42.4, 34.8, 24.1, 22.7, 22.1, 14.2; HRMS (AP-ESI) m/z calcd for
C20H31N6O5 [M+H]+ 435.2356, found 435.2344.
5.1.5.25.
(S)-2-(3-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22x)
1
White solid, yield: 65%, mp: 146.2-148.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.68 (s, 1H), 8.80 (s, 1H), 8.75 (s, 1H), 8.06 (s, 2H), 7.76 (s, 1H), 6.50 (t, J = 5.4 Hz,
1H), 6.19 (d, J = 9.0 Hz, 1H), 4.34-4.32 (m, 2H), 4.08 (q, J = 9.0 Hz, 1H), 1.55-1.51 (m,
13
1H), 1.36-1.34 (m, 2H), 0.88-0.85 (m, 6H); C-NMR (100 MHz, DMSO-d6): δ 169.8,
157.6, 147.8, 138.7, 135.7, 128.3, 128.2, 121.5, 118.9, 118.9, 49.6, 42.8, 35.2, 24.6,
23.2, 22.6; HRMS (AP-ESI) m/z calcd for C16H21Cl2N6O3 [M+H]+ 415.1052, found
415.1045.
5.1.5.26.
(S)-2-(3-((1-(3,4-Dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)ureido)-N-
hydroxy-4-methylpentanamide (22y)
1
White solid, yield: 57%, mp: 178.4-180.2 °C. H NMR (600 MHz, DMSO-d6): δ
10.69 (s, 1H), 8.81 (s, 1H), 8.50 (s, 1H), 7.51 (s, 2H), 7.11 (s, 1H), 6.47 (t, J = 5.4 Hz,
1H), 6.18 (d, J = 8.4 Hz, 1H), 4.33-4.30 (m, 2H), 4.08 (q, J = 8.4 Hz, 1H), 2.36 (s, 6H),
1.55-1.53 (m, 1H), 1.36-1.34 (m, 2H), 0.88-0.85 (m, 6H); 13C-NMR (100 MHz, DMSO-
d6): δ 169.8, 157.7, 147.2, 138.5, 137.3, 135.0, 131.0, 121.2, 120.9, 117.6, 49.6, 42.8,