Synthesis and biological evaluation of novel benzofuroxan-based pyrrolidine hydroxamates as matrix metalloproteinase inhibitors with nitric oxide releasing activity

Article abstract of DOI:10.1016/j.bmc.2018.06.023

On the basis of the strategy of “multifunctional drugs”, a series of novel matrix metalloproteinase inhibitors (MMPIs) containing benzofuroxan scaffold as a nitric oxide donor were designed, synthesized and evaluated. All synthesized compounds, especially 16a, exhibited potent MMP-2,9 inhibitory activities, anti-proliferative activities and could produce high levels of NO in Hela cells. They were also evaluated for both of their anti-invasion and anti-angiogenesis effects. Furthermore, compared with LY52, 16a demonstrated competitive antitumor activity in vivo. These hybrid NO-MMPIs might offer suitable scaffolds to develop valuable MMP inhibitors for the further discovery of novel anti-cancer drugs.

Full text of DOI:10.1016/j.bmc.2018.06.023

Accepted Manuscript
Synthesis and biological evaluation of Novel benzofuroxan-based pyrrolidine
hydroxamates as matrix metalloproteinase inhibitors with nitric oxide releasing
activity
Hao Zhang, Xuejian Wang, Jing Mao, Yongxue Huang, Wenfang Xu, Yu Duan,
Jian Zhang
PII:
S0968-0896(18)30399-7
https://doi.org/10.1016/j.bmc.2018.06.023
BMC 14415
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
24 February 2018
17 May 2018
15 June 2018
Please cite this article as: Zhang, H., Wang, X., Mao, J., Huang, Y., Xu, W., Duan, Y., Zhang, J., Synthesis and
biological evaluation of Novel benzofuroxan-based pyrrolidine hydroxamates as matrix metalloproteinase inhibitors
with nitric oxide releasing activity, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.06.023
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Synthesis and biological evaluation of Novel benzofuroxan-based
pyrrolidine hydroxamates as matrix metalloproteinase inhibitors
with nitric oxide releasing activity
Hao Zhang1, Xuejian Wang2, Jing Mao1, Yongxue Huang3, Wenfang Xu4, Yu Duan1*,
Jian Zhang1*
1Department of Medicinal Chemistry, School of Pharmacy, Weifang Medical
University, Weifang, Shandong 261053, P.R. China
2Department of Pharmacology, School of Pharmacy, Weifang Medical University,
Weifang, Shandong 261053, P.R. China
3Weifang Bochuang International Biological Medicinal Institute, Weifang, Shandong,
261061, P.R. China
4Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong
University, Ji’nan, Shandong 250012, P.R. China
*Address correspondence to this author at the Department of Medicinal Chemistry, School of
Pharmacy, Weifang Medical University, Weifang, Shandong 261053, China; Tel:+86-536-
8462065 ;Fax:+86-536-8462539; E-mail: duanyu@wfmc.edu.cn； zhangjian_3323@163.com
ABSTRACT
On the basis of the strategy of “multifunctional drugs”, a series of novel matrix
metalloproteinase inhibitors (MMPIs) containing benzofuroxan scaffold as a nitric
oxide donor were designed, synthesized and evaluated. All synthesized compounds,
especially 16a, exhibited potent MMP-2,9 inhibitory activities, anti-proliferative
activities and could produce high levels of NO in Hela cells. They were also evaluated
for both of their anti-invasion and anti-angiogenesis effects. Furthermore, compared
with LY52, 16a demonstrated competitive antitumor activity in vivo. These hybrid
NO-MMPIs might offer suitable scaffolds to develop valuable MMP inhibitors for the
further discover of novel anti-cancer drugs.
Key words: MMP inhibitor, nitric oxide, benzofuroxan, multifunctional drugs,
synthesis.
1. Introduction
The matrix metalloproteinases (MMPs) are a family of structurally and functionally
related zinc-dependent endoproteases that are involved in the degradation and
remodeling of the extracellular matrix (ECM).1 MMPs have been linked to

physiological and pathological processes such as cancer growth, arthritis,
inflammation and cardiovascular.2-4 Among all the identified MMP subtypes, MMP-2
(Gelatinases A) and MMP-9 (Gelatinases B), have been implicated in virtually all
aspects of cancer progression (angiogenesis, invasion and metastasis), making them
important targets for intervention. 5
Nitric oxide (NO) is a key mediator that plays very important roles in mammalian
physiology and pathophysiology. High levels of NO and its metabolic derivatives, the
reactive nitrogen species (RNS) and reactive oxygen species (ROS), have profound
effects on tumor cells proliferation, angiogenesis and metastasis by functional
proteins.6 Recently, there is some evidence that the production of NO by some
synthesized NO-releasing compounds has shown potent cytotoxicity against human
carcinoma cells in vitro and anti-metastasis activity in vivo. 7, 8 Indeed, exogenous
NO-donors, such as furoxans, have shown stable against acid and base, and it can
produce high levels of NO, leading to potent cytotoxicity against broad types of
cancer cells.
Previous studies have demonstrated that increased NO decreases MMP-2,9
expression and activity. The exact relationship between NO and MMP-2,9 is evidently
complicated. Increased expression of MMP-2 and MMP-9 is responsive to various
cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α).9-11
High-output levels of nitric oxide (NO) is reported to interact with these signaling
pathways by the inducible isoform of NO synthase (iNOS), thus MMP-inhibitory NO
donor hybrid might be useful to downregulate MMP-2,9 expression and it could play
a significant role for the pharmaceutical treatment in pathological situations.12, 13
The current authors' and their colleagues have recently endeavored to identify
pyrrolidine derivatives as effective MMP inhibitors, exemplified by LY52 (Figure
1).14, 15 Vast number of sulfonamide-based derivatives as potential MMP inhibitors
including AG 3340 and RS 130830 have also been reported (Figure 1).16, 17
Accordingly, the introduction of the sulfonamide group and caffeoyl group into the
MMP inhibitors enhanced their zinc-binding effect, and also enable it to plunge into
the enzyme binding domain deeply. Inspired by the theory of ‘multifunctional drugs’,
we thereof have designed and synthesized a series of novel NO-MMPIs containing
18
benzofuroxan as the NO donor, which is integrated at the C-4 position of the
pyrrolidine scaffold. The 4-phenoxyphenylsulfonyl and 3,4-dimethoxyphenylacryloyl
group are introduced into the cis-γ-amino-L-proline scaffold occupying the deep S1′
pocket, resulting effective enzyme interaction.19-21 The hydroxamate was known as an
effective ZBG to chelate the active site of catalytic zinc ions. 22 In the present study,
we hereby described the synthesis and relevant biological evaluation of all the
compounds.

Figure 1. Design strategy of the target compounds.
2. Chemistry
The routes of intermediate compounds 11a and 11b were efficiently synthesized
following the procedures as illustrated in Scheme 1. The key intermediate
(2S,4S)-1-tert-butyl-2-methyl-4-aminopyrrolidine-1,2-dicarboxylate (6) was prepared
by esterification, acylation, sulfonation, SN2 nucleophilic substitution, and Staudinger
reaction.23 The intermediate 5(6)-carboxybenzo[c][1,2,5]oxadiazole 1-oxide (9a) was
prepared starting from the 4-amino-3-nitrobenzoic acid (7a) by diazo-reaction with
sodium azide to get compound 8a, then refluxed in toluene. The whole preparation
processes of target compounds 9b were similar as that of 9a. The target compounds
16a and 16b were prepared via the synthetic route showed in Scheme 2. The
intermediate compound 11a and 11b were treated with 4-Phenoxybenzene-1-sulfonyl
chloride (12) to get compounds 14a and 14b, which be treated with NH2OK in
anhydrous methanol to yield the target compounds 16a and 16b.
The target compounds 17a and 17b were prepared via the synthetic route showed in
Scheme 2. The intermediate compound 11a and 11b were treated with
(E)-3-(3,4-Dimethoxyphenyl)-2-propenoic acid (13) to get compounds 15a and 15b,
which be treated with NH2OK in anhydrous methanol to yield the target compounds
17a and 17b.

Scheme 1. Synthesis route of intermediate 11a and 11b. Reagents and conditions: (a) 33%
NaNO2, 35% HCl, NaN3, 84%; (b) toluene, 80%; (c) IBCF, THF, 76%; (d) TFA, DCM, 92%.
Scheme 2. Synthesis route of compounds 16a, 16b, 17a and 17b. Reagents and conditions: (a)
ClSO2OH, DCM, SOCl₂, 89%; (b) THF, 72%; (c) Me2SO4, NaOH, 78%; (d) IBCF, THF, 61%; (e)
NH2OK, MeOH, 66%.
3. Results and discussion
3.1. MMP-2 and MMP-9 inhibition assay

The newly synthesized benzofuroxan pyrrolidine derivatives were assayed for their
inhibitory activities toward MMP-2 and MMP-9, and LY52 served as the positive
control. The results of their inhibitory activities (IC50) were reported in Table 1, the
target compounds (16a, 16b, 17a and 17b) displayed the potent inhibitory activity
toward MMP-2 and MMP-9. Notably, all the compounds displayed better inhibitory
activities against MMP-9 compared with MMP-2. Moreover, compound 16a and 16b
with 4-phenoxyphenylsulfonyl group were more potent than the others and control
LY52, among which 16a possessed the most potent activity in nanomole level. This
result revealed that the inhibitory activities of MMPs were affected dramatically by
the characteristics of S1′ pocket group, in addition, the compounds at the
meta-position of the benzofuroxan were more potent than those of being at the
ortho-position.
Table 1 The inhibitory activities of the target compounds (16a, 16b, 17a and 17b)
against MMP-2, MMP-9.
Compd
IC50 (nM)a
MMP-2
102±31.4
182±25.2
345±31.4
414±21.3
266±19.2
MMP-9
162±10.5
242±29.2
388±18.7
524±39.6
360±30.1
16a
16b
17a
17b
LY52
a Results expressed as the mean ± standard deviation of at least three separate determinations.
3.2. In vitro antiproliferation assays
Compounds 16a, 16b, 17a and 17b were evaluated for their antiproliferative
activities against different tumor cell lines of A549 (lung cancer cell), ES-2(ovarian
clear cell carcinoma cell), Hela (human cervical carcinoma cell), K562 (chronic
myelogenous leukemia cell), MCF-7 (human breast adenocarcinoma cell), and
MDA-MB-231 (human breast adenocarcinoma cell), whereas LY52 were used as
positive control. The results presented in Table 2 demonstrated that the effect of all
compounds against Hela and K562 cells were better than those against the other cells.
Compound 16a exhibited the most potent antiproliferative activities, which was
consistent with the previous results of enzyme inhibition.

To confirm the further anti-growth activity of synthesized compounds, the colony
formation assay was performed to measure the effect of 16a, 16b, 17a and 17b in
Hela cells. As shown in Figure 2, all compounds could lead to a significant reduction
in the number of Hela cell colony formation in comparison with the control group.
Furthermore, 16a and 17a showed better potency than LY52 at the same
concentration.
Table 2 The inhibitory activities of compounds 16a, 16b, 17a, 17b and LY52 against
several tumor cells.
Compd
IC50 (μM)a
A549
ES-2
Hela
K562
MCF-7
MDA-MB-231
16a
5.10±0.49 7.47±4.11 3.82±0.11
21.49±0.31 8.26±3.01 7.24±0.22
13.23±1.93 14.11±2.21 6.49±0.76
3.56±0.15 4.19±0.65
4.58±1.73 12.96±0.37
4.61±0.74 10.66±1.90
7.01±1.02
4.19±0.64
9.81±0.11
16.49±1.48
>200
16b
17a
17b
LY52
25.64±1.37 17.35±1.33 14.07±1.98 11.24±1.32 23.45±0.48
78.25±5.6 >200 184.62±8.4 >200 >200
a Results expressed as the mean ± standard deviation of at least three separate determinations.
A
Control
LY52
16a
16b
17a
17b

B
Figure 2. Colony formation of Hela cells observed under an inverted microscope (A). The
inhibition rate was estimated by counting the number of colonies containing more than 50 cell (B).
3.3. Cell migration assay
Cell migration and invasion are essential processes which is correlated with tumor
metastasis. The migration of Hela cells was assessed using a wound healing assay.
The result of the wounded area we found that 16a effectively inhibited cell migration
with a treatment duration of 0–48 h in a time-dependent manner (Figure 3).
A
16a
LY52

0 h
12 h
16a
LY52
24h
48 h
B
Figure 3. Cells migration were treated with 1 μM 16a by the wound healing assay, imaging (A)
and wound closure (B) of migrated cells.
3.4. Cell invasion assay
The invasion of Hela cells was assessed using Transwell chambers. The results
show that compounds 16a, 16b, 17a and 17b significantly inhibited cellular invasion
(Figure 4) in Hela cells than the control group. Furthermore, 16a displayed better
inhibitory activity on Hela cells migration and invasion than LY52.
A

Control
LY52
16a
16b
17a
17b
B
Figure 4. Anti-invasion activity of compounds against Hela cells at 1 μM, imaging (A) and cell
number (B) of migrated cells.
3.5. HUVECs tuber formation assay
Before studying the anti-angiogenic effects of the target compounds, we first
evaluated the cytotoxicities of these compounds against human umbilical vein
endothelial cells (HUVECs) by the MTT assay. All the tested compounds (16a, 16b,

17a, 17b and LY52) demonstrated low cytotoxic activities with CC50 values of 32.53
μM, 37.17 μM, 36.26 μM, 42.33 μM and 39,84 μM, respectively.
HUVECs tube formation assay was then performed in vitro. Results in Figure 5
showed that all the tested compounds significantly suppressed tube formation of
HUVEC compared with the blank control, and the inhibition rate of 16a was much
higher than that of LY52 at the same concentration.
A
Control
LY52
16a
16b
17a
17b
B

Figure 5. The tube formation assay depicting the formation of a HUVEC capillary-like tubular
network by 1 μM treatment with 16a, 16b, 17a, 17b, LY52 (positive control) and vehicle control.
The images were captured by an inverted microscope (A). Quantification of tube formation (B).
3.6. Rat thoracic aorta rings (TARs) assay
Compared with HUVECs tuber formation, rat aortic thoracic aorta rings assay
model is more close to in vivo condition involved in process of angiogenesis. All the
synthesized compounds were performed to evaluate the microvessel formation at the
concentration of 5 μM. As shown in Figure 6, both 16a, 17a and LY52 could
suppress angiogenesis formation than the control. Moreover, 16a and 17a showed
outstanding angiogenesis inhibition ratio than LY52, while 16b and 17b turned up to
be moderate or weak ones.
A
Control
LY52
16a

16b
17a
17b
B
Figure 6. Rat aortic rings assay treated with 16a, 16b, 17a, 17b, LY52 (positive control) and
vehicle control at 5 μM. The images were captured by an inverted microscope (A). The inhibition
rates on microvessel outgrowth arising from the rat aortic ring were counted (B).
3.7. NO generation measurement
The levels of the NO generated by compounds 16a, 16b, 17a and 17b were
determined and presented as that of nitrite using a Griess assay.24 Hela cells were
respectively exposed to 100 μM of each compound in the same incubation time 5
hours. All the tested compounds could release NO efficiently (Figure 7). Especially,
compound 16a released considerable amount of NO in 34.43 μM/L, while the less
active compounds 16b, 17a and 17b released lower levels of NO (27.87, 33.11, 25.51
μM, respectively).

Figure 7. The levels of NO released by target compounds were determined.
3.8. In vivo antitumor activity (16a extended lifespan of tumor bearing mice)
The lifespan extension effects of 16a on H22-bearing KM mice were examined by
comparing the mean survival time of each mice group. As shown in Figure 8,
compound 16a and LY52 displayed longer mice survival times when compared with
the vehicle control. Furthermore, compound 16a was observed to enhance the survival
rate to about 40.48%. During the evaluation of compounds, the weight of mice in the
control group and the positive group were increased, while the weight of mice in 16a
group was gradually reduced, which may be related to some toxicity of compound.
Figure 8. Survival curves of tumor bearing mice received different treatments.
3.9. Molecular docking
Compound 16a was chosen to be carried out docking studies for investigating its
binding mode in the active site of MMP-2 (PDB: 1HOV). The result showed in

Figure 9 suggested that the hydroxamic acid group can chelate with the catalytic zinc
ion and the arylsulfonyl group can insert into S1’ pocket, while, the benzofuroxan
group can occupy S2’ pocket. For a detailed understanding of the interactions of 16a
with the residues lined in the active site of the protein, the picture of H-bond
interactions was also created (Figure 10). The benzofuroxan group of compound 16a
can form hydrogen bonds with Gly81 and Tyr112 of S2’ pocket and the carbonyl group
of compound 16a can form hydrogen bonds with His124 and His130.
Figure 9. The FlexX Docking of Compound 16a with MMP-2
Figure 10. The binding mode of Compound 16a with MMP-2
4. Conclusions
In summary, several novel benzofuroxan pyrrolidine derivatives against MMPs
were designed, synthesized and evaluated. All these hybrid compounds showed more
potent inhibitory activities against MMP-2 and MMP-9 than the positive control
LY52. One representative compound 16a displayed significant in vitro
anti-proliferation, anti-metastasis, anti-angiogenesis and could release high levels of

NO. Moreover, compound 16a exhibited a comparable lifespan extension effects in
vivo compared to LY52. Collectively, these results suggest that 16a could be used as a
promising candidate for further structural optimization and evaluation to develop
MMPs inhibitors in the future.
5. Experimental sections
5.1. Chemistry
Unless otherwise noted, all the materials including reagents and solvents were
commercially available and used without further purification. All reactions were
monitored by TLC with 0.25 mm silica gel plates (60GF-254) and visualized with UV
light, iodine stain, and ferric chloride. Silica gel or C18 silica gel was used for column
1
chromatography purification. H NMR and 13C NMR spectra were recorded on a
Bruker DRX spectrometer at 600 MHz, d in parts per million and J in hertz, using
TMS as an internal standard. HR-MS spectra were determined on an Agilent
Q-TOF-6250 spectrometer at the Shandong Analysis and Test Center in Ji'nan, China.
Melting points were determined on an electrothermal melting point apparatus.
The compound 6 were synthesized following previously reported procedures.23
5(6)-Carboxybenzo[c][1,2,5]oxadiazole 1-oxide (9a)
To a solution of 4-Amino-3-nitrobenzoic acid (3.6 g, 20 mmol) in 100 mL 35% (v/v)
HCl at ice bath atmosphere (0–5 °C), 4.4 mL 33% sodium nitrite solution was added
slowly. After stirring for 1 h at 0–5 °C, sodium azide solution (1.3 g in 5 mL H2O)
was dropwise added into the slurry and keeping the inner temperature lower than
15 °C. Additional 1.5 h stirred later, the mixture was filtered and the filter cake was
washed with saturated sodium hyposulfite. After vacuum dried, the compound 8a was
obtained as yellow solid. Then, the compound 8a was resolved in 100 mL toluene and
refluxed at 80 °C for another 1 h. The mixture was filtered to get yellow solid, which
was recrystallized with ethanol to give the compound 9a as yellow solid, yield 80%.
1H NMR (600 MHz, DMSO-d6) δ 13.80 (s, 1H), 8.21 (d, J = 182.0 Hz, 1H), 7.86 (s,
2H)
4(7)-Carboxybenzo[c][1,2,5]oxadiazole 1-oxide (9b)
The method is as the same as the preparation of compound 9a. yellow solid, yield
1
78%, H NMR (600 MHz, DMSO-d6) δ 13.94 (s, 1H), 8.13 (d, 2H), 7.52 (t, J = 7.9,
1.3 Hz, 1H).
5(6)-(((3S,5S)-1-(Tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidin-3-yl)carb
amoyl)benzo[c][1,2,5]oxadiazole 1-oxide (10a)
On an ice bath, to a solution of 9a (2.2 g, 12.2 mmol) in anhydrous THF (30 mL), was
added Et3N (3.7 g, 36.6 mmol) followed by Isobutyl chloroformate (2.0 g, 14.66
mmol). After 15 min, the compound 6 (4.11 g, 14.66 mmol) was added. After reaction,
THF was evaporated with the residue being taken up in EtOAc (50 mL). The EtOAc
solution was washed with saturated aqueous citric acid, saturated aqueous NaHCO3,

and brine, dried over MgSO4 and evaporated to give compound 10a as yellow oil,
76%. 1H NMR (600 MHz, DMSO-d6) δ 8.80 (d, J = 6.7 Hz, 1H), 8.13 (s, 1H), 7.80 (s,
1H), 4.44 (t, J = 7.3 Hz, 1H), 4.29 (q, 1H), 3.78 (d, J = 8.8 Hz, 1H), 3.74 (d, J = 3.2
Hz, 1H), 3.68 (s, 3H), 2.59 (d, J = 6.8 Hz, 1H), 2.07 (d, J = 7.5 Hz, 1H), 1.35 (s, 9H).
4(7)-(((3S,5S)-1-(Tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidin-3-yl)carb
amoyl)benzo[c][1,2,5]oxadiazole 1-oxide (10b)
1
The method is as the same as the preparation of compound 10a. yellow oil, 76%, H
NMR (600 MHz, DMSO-d6) δ 8.93 (d, J = 6.5 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.75
(t, J = 8.0, 1.9 Hz, 1H), 4.43 (q, J = 7.0 Hz, 1H), 4.29 (t, J = 7.5 Hz, 1H), 3.80 (d, J =
10.3 Hz, 2H), 3.69 (s, 3H), 3.30 (s, 1H), 2.62 (s, 1H), 1.41 (s, 9H).
6-(((3S,5S)-5-(Methoxycarbonyl)pyrrolidin-3-yl)carbamoyl)benzo[c][1,2,5]oxadia
zole 1-oxide (11a)
On an ice bath, 10a or 10b (10 mmol) was added to a solution of trifluoroacetic acid
and dichloromethane (1:1, 10 mL). The mixture was reacted for 5 h and TLC was
used to monitor completion. The solvent was evaporated to give yellow oil, 91%. 1H
NMR (600 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.59 (s, 1H), 9.32 (d, J = 27.6 Hz, 1H),
7.90 (d, J = 23.3 Hz, 1H), 4.65 – 4.59 (m, 1H), 4.54 (t, J = 8.9 Hz, 1H), 3.78 (s, 3H),
3.53 – 3.49 (m, 1H), 3.39 – 3.33 (m, 1H), 2.69 (dt, J = 13.6, 8.2 Hz, 1H), 2.25 (dt, J =
14.6, 7.9 Hz, 1H).
7-(((3S,5S)-5-(Methoxycarbonyl)pyrrolidin-3-yl)carbamoyl)benzo[c][1,2,5]oxadia
zole 1-oxide (11b)
The method is as the same as the preparation of compound 11a. yellow oil, 92%.1H
NMR (600 MHz, DMSO-d6) δ 10.77 (s, 1H), 9.62 (s, 1H), 9.34 (d, J = 6.4 Hz, 1H),
8.08 (d, J = 8.1 Hz, 1H), 4.56 (dt, J = 21.6, 7.4 Hz, 1H), 3.77 (s, 3H), 3.52 (t, J = 10.0
Hz, 1H), 3.38 – 3.31 (m, 1H), 2.74 – 2.66 (m, 1H), 2.20 (dt, J = 14.4, 7.9 Hz, 1H),
1.91 (m, 1H).
4-Phenoxybenzene-1-sulfonyl chloride (12)
In a 25 mL round-bottom flask, diphenyl ether (1.1 mg, 6.46 mmol) was dissolved in
dry DCM (30 mL), and chlorosulphonic acid (0.78 mL, 6.46 mmol) was added at 0 °C.
The reaction was stirred for 2 h, evaporated under vacuum, and used for the next step
without further purification. The reaction mixture was indeed dissolved in thionyl
chloride at 0 °C and refluxed for 5 h to yield after evaporation product 12 with 89%
yield. 1H NMR (600 MHz, DMSO-d6) δ 7.65 – 7.55 (m, 2H), 7.23 (q, J = 7.8 Hz, 2H),
6.99 (m, J = 7.6 Hz, 1H), 6.88 (t, J = 8.8 Hz, 2H), 6.84 (t, J = 9.2 Hz, 2H).
(E)-3-(3,4-Dimethoxyphenyl)-2-propenoic acid (13)
Caffic acid (18 g, 0.1 mol) was dissolved in cool 90 mL NaOH solution (4mol/L),in
which process the inner temperature was required under 20 °C. After Me2SO4 (20 mL)
was added dropwise and stirred for 20 min, 50 mL NaOH solution (4 mol/L) and 20
mL Me2SO4 were dropped into the reaction system constantaneously. The resulting

mixture was slowly heated to 90 °C in 1 h and maintained at this temperature for 1 h,
then the solution was refluxed for 2 h, after 50 mL NaOH solution (4 mol/L) was
added, another 2 h of refluxing was applied. The pH of the resulting solution was
regulated to 2 by concentrated HCl. Two to three hours later, mass brown solid were
attained by filtrating, and washed to neutrality with water. The gross was
1
recrystallized in EtOH and H2O to get 16.4 g (yield 78%) light yellow crystal. H
NMR (600 MHz, DMSO-d6) δ 12.21 (s, 1H), 7.53 (d, J = 15.9 Hz, 1H), 7.32 (s, 1H),
7.21 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.45 (dd, J = 15.9, 2.3 Hz, 1H),
3.80 (dd, J = 8.9, 2.3 Hz, 6H).
5(6)-(((3S,5S)-5-(Methoxycarbonyl)-1-((4-phenoxyphenyl)sulfonyl)pyrrolidin-3-y
l)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide (14a)
On an ice bath, 10a (10 mmol) was added to a solution of trifluoroacetic acid and
dichloromethane (1:3, 40 mL). The mixture was reacted for 1 h and TLC was used to
monitor completion. The solvent was evaporated to give brown oil, which was
dissolved in saturated NaHCO3 solution to pH 7. After extraction with
dichloromethane, the organic layer was evaporated to give a secondary amine as
brown oil, which was used for the subsequent reaction without further purification. At
room temperature, to a solution of the secondary amine (0.71 g, 2.3 mmol) in
anhydrous THF (25 mL) was added Et3N (0.46 g, 4.6 mmol), and the compound 12
(0.62 g, 2.3 mmol) was added at 0 °C. Stirring was continued for 1 h, and then THF
was evaporated with the residue being taken up in EtOAc (50 mL). The EtOAc
solution was washed with saturated Na2CO3 (3 ×10 mL) and brine (3×10 mL), dried
over MgSO4, and evaporated under vacuum. The crude product was purified by flash
column chromatography (petroleum ether/EtOAc 2:1) to give desired compound 14a
(0.85 g, 80% yield) as a yellow oil. 1H NMR (600 MHz, DMSO-d6) δ 8.83 (d, J = 6.2
Hz, 1H), 8.11 (s, 1H), 7.90 – 7.86 (m, 2H), 7.78 (s, 1H), 7.52 – 7.47 (m, 2H), 7.28 (t,
J = 7.5 Hz, 1H), 7.24 – 7.16 (m, 4H), 4.35 (t, J = 7.7 Hz, 1H), 4.11 (q, J = 7.1 Hz, 1H),
3.69 (s, 3H), 3.61 (m, 2H), 2.47 (d, 1H), 2.11 (d, 1H).
4(7)-(((3S,5S)-5-(Methoxycarbonyl)-1-((4-phenoxyphenyl)sulfonyl)pyrrolidin-3-y
l)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide (14b)
The method is as the same as the preparation of compound 14a. yellow oil, 7
1
2%, H NMR (600 MHz, DMSO-d6) δ 8.96 (d, J = 6.2 Hz, 1H), 8.08 (d, J =
8.1 Hz, 1H), 7.89 (dd, J = 8.8, 2.0 Hz, 2H), 7.74 (d, J = 7.6 Hz, 1H), 7.50
(d, J = 1.9 Hz, 2H), 7.28 (t, J = 7.5 Hz, 1H), 7.22 – 7.14 (m, 4H), 4.37 (t,
J = 7.4 Hz, 1H), 4.14 (q, J = 6.9 Hz, 1H), 3.70 (d, J = 1.9 Hz, 3H), 3.62
(dd, J = 10.2, 7.6 Hz, 2H), 2.51 (d, 1H), 2.08 (d, J = 13.3, 6.8 Hz, 1H).
5(6)-(((3S,5S)-1-((E)-3-(3,4-Dimethoxyphenyl)acryloyl)-5-(methoxycarbonyl)pyrr
olidin-3-yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide (15a)
At 0 °C, to a solution of 13 (0.87 g, 4.2 mmol) in anhydrous THF (25 mL) was added
Et3N (1.06 g, 10.5 mmol) followed by Isobutyl chloroformate (0.63 g, 4.62 mmol).
After 15 min, the amine 11a (1.42 g, 4.62 mmol) was added. Stirring was continued

for 3 h, and then THF was evaporated with the residue being taken up in EtOAc (40
mL). The EtOAc solution was washed with saturated Na2CO3 (3×10 mL) and brine
(3×10 mL), dried over MgSO4, and evaporated under vacuum. The crude product was
purified by flash columnchromatography (petroleum ether/EtOAc 1:1) to give desired
1
compound 15a (1.3 g, 61% yield) as a yellow oil. H NMR (600 MHz, DMSO-d6) δ
8.93 (d, J = 6.9 Hz, 1H), 8.16 (s, 1H), 7.86 (s, 1H), 7.45 (d, J = 15.4 Hz, 1H), 7.38 (d,
J = 2.0 Hz, 1H), 7.22 (d, J = 1.9 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 15.4
Hz, 1H), 4.63 (q, J = 7.4 Hz, 1H), 4.49 (t, J = 8.0 Hz, 1H), 4.28 (dd, J = 10.3, 7.1 Hz,
1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.66 (s, 3H), 3.62 (d, J = 9.4 Hz, 1H), 2.60 (d, 1H),
2.06 (d, 1H).
4(7)-(((3S,5S)-1-((E)-3-(3,4-Dimethoxyphenyl)acryloyl)-5-(methoxycarbonyl)pyrr
olidin-3-yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide (15b)
1
The method is as the same as the preparation of compound 15a. yellow oil, 57%, H
NMR (600 MHz, DMSO-d6) δ 9.01 (d, J = 6.7 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.77
(dd, J = 7.8, 1.9 Hz, 1H), 7.46 (d, J = 15.3 Hz, 1H), 7.38 (s, 1H), 7.26 – 7.21 (m, 1H),
6.98 (d, J = 8.4 Hz, 1H), 6.92 (d, 1H), 4.61 (q, J = 7.0 Hz, 1H), 4.51 (t, J = 7.9 Hz,
1H), 4.27 (d, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 3.70 (s, 3H), 3.67 (d, 1H), 2.62 (d, 1H),
2.02 (d, 1H).
5(6)-(((3S,5S)-5-(Hydroxycarbamoyl)-1-((4-phenoxyphenyl)sulfonyl)pyrrolidin-3-
yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide（16a）
To a solution of compound 15a (1.0 g, 2.0 mmol) in 10 mL of anhydrous methanol
was added a solution of NH2OK (16 mmol) in 10 mL of anhydrous methanol. The
mixture was stirred for 0.5 h, and the solvent was evaporated under vacuum. The
residue was acidified with 2 N HCl until pH 5-6 and the mixture was filtered to get
yellow solid. The crude product was purified by flash column chromatography
(petroleum ether/EtOAc 1:1) to give desired compound16a. Yellow solid, 66%, MP:
92-94 °C, 1H NMR (600 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.41 (s, 1H), 9.29 (d, J =
1.9 Hz, 1H), 8.48 (s, 1H), 8.16 (d, J = 9.4 Hz, 1H), 7.92 (m, 2H), 7.48 (m, 2H), 7.28 (t,
J = 7.4 Hz, 1H), 7.18 (m, 4H), 4.24 (q, J = 6.0 Hz, 1H), 4.05 (dd, J = 9.2, 4.6 Hz, 1H),
3.47 (dd, J = 10.6, 5.9 Hz, 2H), 2.31 (d, 1H), 2.01 (d, J = 8.9 Hz, 1H). 13C NMR (151
MHz, DMSO-d6) δ 168.36, 164.24, 161.63, 155.20, 149.56, 149.27, 138.09, 131.47,
130.88, 130.72, 130.53, 125.49, 120.54, 118.26, 117.09, 116.15, 58.42, 54.51, 49.00,
35.84. ESI-MS m/z: 540.1184 [M+H]+.
4(7)-(((3S,5S)-5-(Hydroxycarbamoyl)-1-((4-phenoxyphenyl)sulfonyl)pyrrolidin-3-
yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide （16b）
Yellow solid, 61%, MP: 104-106 °C, 1H NMR (600 MHz, DMSO-d6) δ 11.06 (s, 1H),
9.28 (s, 1H), 9.20 (d, J = 7.5 Hz, 1H), 8.21 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 8.4 Hz,
2H), 7.87 (d, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1H), 7.21 – 7.07 (m,
4H), 4.23 (d, J = 6.0 Hz, 1H), 4.02 (q, 1H), 3.43 (dd, J = 10.7, 5.9 Hz, 2H), 2.51 (d,

1H), 2.28 (d, 1H). 13C NMR (151 MHz, DMSO-d6) δ 168.39, 167.38, 161.62, 155.25,
145.98, 136.04, 132.72, 132.34, 130.89, 130.87, 130.52, 125.50, 120.55, 119.88,
118.26, 114.47, 58.34, 54.74, 48.65, 35.77. ESI-MS m/z: 540.1262 [M+H]+.
5(6)-(((3S,5S)-1-((E)-3-(3,4-Dimethoxyphenyl)acryloyl)-5-(hydroxycarbamoyl)py
rrolidin-3-yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide (17a)
Yellow solid, 64%, MP: 122-124 °C, 1H NMR (600 MHz, DMSO-d6) δ 10.90 9.32 (s,
1H), 9.04 (s, 1H), 8.16 (dd, J = 19.9, 9.6 Hz, 1H), 7.86 (s, 1H), 7.43 (d, J = 15.4 Hz,
1H), 7.36 (d, J = 2.0 Hz, 1H), 7.24 – 7.18 (m, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.91 (d, J
= 15.4 Hz, 1H), 4.65 (q, J = 6.7 Hz, 1H), 4.37 (dd, J = 8.9, 5.2 Hz, 1H), 4.12 (dd, J =
10.5, 6.2 Hz, 2H), 3.81 (s, 3H), 3.79 (s, 3H), 2.67 (d, J = 14.0, 7.7 Hz, 1H), 2.14 (d, J
13
= 12.7, 6.1 Hz, 1H). C NMR (151 MHz, DMSO-d6) δ 169.53, 164.92, 163.69,
150.99, 149.50, 141.97, 141.96, 128.23, 128.14, 123.17, 123.04, 122.62, 117.49,
116.65, 112.27, 112.14, 111.03, 57.07, 56.23, 53.11, 49.39, 47.87, 34.69. ESI-MS m/z:
498.1625 [M+H]+.
4(7)-(((3S,5S)-1-((E)-3-(3,4-Dimethoxyphenyl)acryloyl)-5-(hydroxycarbamoyl)py
rrolidin-3-yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-oxide (17b)
Yellow solid, 60%, MP: 132-135 °C, 1H NMR (600 MHz, DMSO-d6) δ 10.95 (s, 1H),
9.18 (d, J = 7.8 Hz, 1H), 9.07 (s, 1H), 8.28 (d, J = 30.8 Hz, 1H), 8.20 (d, J = 8.6 Hz,
1H), 7.91 (d, J = 7.4 Hz, 1H), 7.65 (dd, J = 12.5, 7.6 Hz, 1H), 7.44 (d, J = 15.4 Hz,
1H), 7.21 (d, J = 8.5 Hz, 1H), 6.98 – 6.91 (m, 1H), 4.67 (q, J = 6.2 Hz, 1H), 4.39 (dd,
J = 9.0, 4.6 Hz, 1H), 4.08 (dd, J = 10.5, 6.1 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 2.68
(d, J = 6.9 Hz, 1H), 2.12 (d, J = 12.1, 6.2 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ
169.61, 164.87, 163.72, 150.93, 149.52, 141.99, 141.87, 128.28, 128.07, 123.22,
123.19, 122.35, 117.56, 116.58, 112.35, 112.06, 111.14, 57.12, 56.64, 53.46, 49.45,
47.96, 34.72. ESI-MS m/z: 498.1698 [M+H]+.
5.2. Biological materials and methods
5.2.1. In vitro MMP-2 and MMP-9 inhibition assay
Recombinant human MMP-2, MMP-9 and the fluorogenic substrate
Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 were all purchased from Enzolife.
Our compounds were assayed for their inhibitory activities against MMP-2 and
MMP-9 using Fluorometric Drug Discovery Kit. The assay buffer was pipetted into
each desired well in the total volume of 100 μl. After equilibrated to 37 °C, 20 μL
enzyme was added. ropriate wells. The plate was incubated for 45 min at 37 °C to
allow inhibit Then 20 μL positive control NNGH and test inhibitors were added to
appor/enzyme interaction. Then 10 μL fluorogenic substrate was added to start
reaction. The resulting solutions were measured in the fluorescent microplate reader,
using Ex/Em=328/420.
5.2.2. MTT assay

Cell proliferation was determined by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-
diphenyl-2H-tertazolium bromide) assay. Briefly, the cell lines were grown in
RPMI1640 medium containing 10% FBS at 37 ℃ in 5% CO2 humidified incubator.
Cells were plated in 96-well plates (5000/well) and cultured for 4 h in complete
growth medium, then treated with various concentrations of tested compounds. The
plates were incubated for an additional 48 h, and then 0.5% MTT was added into each
well. Four hours later, 200 μL DMSO was added and the plate was shaken for 5 min.
Finally, the optical density values were determined using an ELISA reader at 570 nm.
5.2.3. Colony formation
Hela cells were seeded in 6-well plates at a density of 500 cells per well and treated
with compounds for 24 h. Cells were incubated for 2 weeks in the environment of
37 °C and 5% CO2, then the cells in each well were washed twice with PBS, fixed
with methanol for 15 min, and stained with 0.1% crystal violet for 10 min. The
colonies were then photographed and counted under an inverted microscope
(Olympus).
5.2.4. Wound healing assay
Cell migration ability was assessed by the wound healing assay as. 2 × 105 cells
were added to the 6-well plate, cultured to 60-80% confluence A plastic pipette tip
was used to generate a clean wound area across the well when cells grown to 100%
confluence. Cells were allowed to migrate in the medium. The wound was visualized
using the inverted microscope (Olympus).
5.2.5. Cell invasion assay
Migration and invasion assays were performed in a 24-well plates using transwell
chamber (Darmstadt, Germany) with a pore size of 8 μm. For migration assay, 5×104
Hela cells were seeded into the upper chamber of transwells (BD Bioscience, Bedford,
MA). For invasion assay, 1×105 Hela cells were added into the upper chamber
precoated with matrigel (BD Bioscience, Bedford,MA). In both assays, cells per well
with compounds were plated into the upper chamber in RPMI1640 medium
containing 1% FBS. The lower chamber contained 10% FBS as a chemoattractant.
After incubation for 24 h at 37 °C, cells in the upper side of the filter were carefully
removed with cotton swabs. Then the membranes were fixed with methanol for 10min
and stained with 0.5% crystal violet, photographed, and counted under an inverted
microscope (Olympus).
5.2.6. HUVECs tuber formation assay
A 24-well chambers were coated with 100 mL of Matrigel (BD Biosciences,
Bedford, MA) and allowed to gel at 37 °C and 5% CO2 for 30 min. Then, 1×105
HUVECs cells per wells were suspended in growth medium and synthesized
compounds were seeded into the wells. After 8 h of incubation at 37 °C, 5% CO2, The
morphology of HUVECs cell tube formation was captured using the Olympus
inverted microscope.

5.2.7. Rat thoracic aorta rings (TARs) assay
A 96-well chambers were coated with 100 mL of Matrigel (BD Biosciences,
Bedford, MA) and allowed to gel at 37 °C and 5% CO2 for 30 min. Thoracic aortas
were excised from 6-week-old male Sprague Dawley rats. After the perivascular
tissue was carefully removed, the thoracic aortas were approximately cut into 1
mm-long ring, embedded into Matrigel-coated wells. Aorta rings were treated with
different compounds in the wells and incubated at 37 °C and 5% CO2 for 6 days. The
microvessels were visualized using the inverted microscope (Olympus).
5.2.8. In Vitro Nitrite Measurement
The production of NO was measured as the accumulation of nitrite in culture
colorimetrically by the Griess reaction using the colorimetric assay kit (Beyotime,
China), according to the manufacturer’s instruction. Briefly, Hela cells (1×105/well) in
a 24-well plate were incubated with each compound for 5 h at 100 μM concentration.
After centrifugation, 100 µL of cell-free supernatant were incubated with an equal
volume of Griess reagent for 10 min in a 96-well microtitre plate. The resulting
absorbance values at 540 nm were determined by a microtitre plate reader, NaNO2
(Beyotime Biotechnology) at different concentrations was prepared for the standard
curve.
5.2.9. Survival of tumor-bearing mice assay
Each Kunming mouse (female, weight 20±2 g, 4-6 week old, 30 mice were divided
randomly into three groups with 10 mice per group. A: control group (PBS with 5%
DMSO); B: LY52 (100 mg/kg/day); and C: 16a (100 mg/kg/day)) were inoculated
subcutaneously with injections of 4 × 107/mL H22 cells on day 0. Each mouse was
treated with drug solution by intragastric administration for ten days once daily. The
weights of the mice were documented before each administration. The mice in each
group were observed daily to monitor their survival to determine the in vivo antitumor
effects of target compound.
5.2.10. Computational-docking assay
The docking study was carried out as follows: the selected compound was
constructed with a Sybyl/Sketch module and optimized using Powell Energetic
Gradiet method with a Tripos force field with the convergence criterion set at 0.05
kcal/mol Å, and assigned with Gasteiger-Hückel method. The docking study of the
selected compound with the active site of MMP-2 was performed using Sybyl/ FlexX
module. The active site was defined as 10.0 Å radius circles around Zn2+ (PDB:
1HOV).
Acknowledgements
This work was supported by Natural Science Foundation of Shandong Province
(Grant No. BS2015YY016), Higher Educational Science and Technology Program of
Shandong Province (Grant No. J17KB098), National Natural Science Foundation of

China (Grant No. 81501683 and 31600386) and Doctoral Foundation of Weifang
Medical University.
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