Synthesis and molecular simulation study of furoic peptidomimetic derivatives as potent aminopeptodase N inhibitors

Article abstract of DOI:10.1691/2018.7911

The aminopeptidase N (APN) plays a critical role in angiogenesis and is over-expressed in tumor cells. In this paper, we report the synthesis and enzyme inhibition assay of furoic peptidomimetic compounds. These new compounds exhibit potent inhibitory ability toward APN with IC₅₀ values lying in the micromolar level. The binding mode of inhibitors in APN active site was explained by a molecular simulation study. These data reveal that ligand coordinating with the catalytic Zn-ion is very important for inhibitory activities.

Full text of DOI:10.1691/2018.7911

ORIGINAL ARTICLES
Department of Medicinal Chemistry1, School of Pharmaceutical Science, NanChang University; People’s Hospital of TongGu
County2, YiChun, JiangXi, China
Synthesis and molecular simulation study of furoic peptidomimetic deriva-
tives as potent aminopeptodase N inhibitors
1
2
1
1
1
1,
MIN GAO , JUNHUA HE , WEIDONG XU , XIAOPING LAI , FEN LIU , GUOGANG TU *
Received October 10, 2017, accepted November 14, 2017
*Corresponding author: GuoGang Tu, Department of Medicinal Chemistry, School of Pharmaceutical Science,
NanChang University, 330006, China
tugg110@163.com
Pharmazie 73: 123–127 (2018)
doi: 10.1691/2018.7911
The aminopeptidase N (APN) plays a critical role in angiogenesis and is over-expressed in tumor cells. In this
paper, we report the synthesis and enzyme inhibition assay of furoic peptidomimetic compounds. These new
compounds exhibit potent inhibitory ability toward APN with IC50 values lying in the micromolar level.The binding
mode of inhibitors in APN active site was explained by a molecular simulation study. These data reveal that
ligand coordinating with the catalytic Zn-ion is very important for inhibitory activities.
to produce compounds 8a-8u (Lassen et al. 2010). The 2-furoic acid
9 was reacted with SOCl2 to obtain furoyl chloride 10 which without
further purification was directly condensed with compounds 8a-8u
to get compounds 11a-11u as the target compounds.
1. Introduction
Aminopeptidase N (APN), as an ectoenzyme, is located on cell
surface (Luan et al. 2007). It is a zinc-dependent metalloprotease
and a type II membrane bound metalloprotease which is widely
expressed in many tissues, such as epithelial cells of the kidney,
fibroblasts, brain cells, intestine and liver (Breljak et al. 2003;
Piela-Smith et al. 1995). The over-expression of APN is associ-
ated with many diseases, such as inflammation, viral infection and
cancer. Especially it plays an important role in tumor metastasis
and angiogenesis (Inagaki et al. 2010). The over-expressed APN
tumor cells (such as melanoma cells and urological cancer cells)
are highly motile and capable of migration through extracellular
matrix (Ishii et al. 2001). Many efficient APN inhibitors have been
reported, such as bestatin, probestin, and lapstatin (Repic Lampret
et al. 1999).
Based on the analysis of the structural characteristic of the APN
active site, it was shown that a zinc binding group (ZBG), two
hydrophobic groups which can occupy the S1 and S ′ pocket and an
appropriate group which can occupy the S2′ poc1ket are usually
necessary (Ito et al. 2006; Addlagatta et al. 2006). APN shows
a broad specificity toward alanine, leucine or arginine when it
preferentially releases the N-terminal neutral and basic amino
acids. Furoic acid and thiadiazole derivatives have been known to
exhibit broad biological properties, especially anti-tumor activities
(Rzeski et al. 2007; Zeng et al. 1998). Based on the ‘combination
principles’, the 2-furoic acid, alanine/leucine and thiadiazole were
linked. A series of novel furoic peptidomimetics were synthesized
and evaluated for their inhibitory activities toward APN. In addi-
tion, the binding mode of the target compounds with the APN
binding site was discussed relying on docking studies, molecular
dynamics (MD) simulation experiments and binding free energies
calculation.
Scheme: Reagents and conditions: (a) POCl3, 110°C; (b) Et3N, r.t. (c) N, N’-carbon-
yldiimidazole, r.t.
(d) trifluoroacetic acid, r.t. (e) refluxed (f) Et3N, r.t.
2.2. Activity testing
All the inhibition results are summarized in the Table. The results
showed that most of the target compounds display moderate
potency toward APN with IC50 values in the micromolar range.
Comparing compounds 11a–11l to 11m-11u, we could confirm that
isobutyl group introduced at the R4-position was negatively related
with the inhibitory activities. The steric effect of the substituent
at the R1-position is extremely important for potency. Compounds
with a tert. butyl group (11f, 11r) exhibited lower activity than
compounds with a methyl group (11d, 11p). For compounds
11a-11c, 11l and 11m-11o introduction of a electron-withdrawing
group slightly increased the activity, while electron-donating groups
remarkably increased the activity. The compounds with a methoxy
group (11e, 11q) showed the best inhibitory activity.
2. Investigations, results and discussion
2.1. Synthesis of the compounds
The target compounds were synthesized efficiently following
the procedures shown in the Scheme. In presence of phosphorus
oxychloride, the readily available carboxylic acid 1 was condensed
with N-aminothiourea 2 to yield the 5-substituted-1,3,4-thiadiazol-
2-amines 3a-3l (Foroumadi et al. 1999). The amino group of amino
acids 4 was protected with di-tert-butyl dicarbonate 5 to get Boc-
amino acids 6a-6b which were coupled with compounds 3a–3l to led
to compounds 7a-7u. Then the Boc protecting group was removed
Pharmazie 73 (2018)
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ORIGINAL ARTICLES
Table 1: In vitro enzyme assay results for compounds 11a-11u and
bestatin
subsites of APN. The thiadiazole nitrogen of compound 11e coordi-
nates with the catalytic Zn-ion with the distance of 2.16 Ǻ, and forms
a hydrogen bond with polar H atom of His297. At the same time, the
hydrophobic parts of aromatic rings are in contact with nonpolar
surface areas of APN. In addition the catalytic Zn-ion coordinates
with His297, His301 and Glu320, which is the same with the crystal
structure of 2DQM. But compound 11m only forms hydrophobic
interaction and could not coordinate with the catalytic Zn-ion. So the
affinity of 11e with APN is higher than that of 11m, which leads to
the better bioactivity of 11e compared to 11m.
To further identify the critical amino acid residues for ligand
binding, MD study and binding free energy calculation were
performed for two docked APN-ligand complexes. The RMSD
values were calculated with 5 ns trajectory to evaluate the stable
binding of compounds 11e and 11m with APN. The RMSD values
of APN backbone atoms and 11e were about 0.1 nm and 0.04 nm,
while the corresponding values of APN-11m complex were about
0.12 nm and 0.1 nm respectively. The both complexes showed a
steady pattern after 2 ns. It can be seen that APN-11e complex is
more stable than the APN-11m complex.
Among the complexes 11e has a better free energy (-190.1 kJ/mol)
binding than 11m (-143.9 kJ/mol) as shown in Table 2. The
energy difference is mainly contributed by electrostatic energy as
well as polar solvation energy. The binding free energy between
APN and ligands was further decomposed into the contribution
of each residue using MM-PBSA approach (Fig. 2). The energy
decomposition analysis shows that the main contributions of 11e
are residues Met260, Val294, Asp327, Tyr376, Tyr381 and Zn900,
while the main contributions of 11m are residues Met260, Glu298,
Glu320, Asp327, Tyr376, and Tyr381. It was shown that Glu298
and Glu320 are in disfavor with the binding for 11e, and Glu121
and Arg293 are in disfavor for 11m. Especially the contribution
of the catalytic Zn-ion is in favor for 11e but disfavor for 11m,
which leads to 11e having more inhibitory activity than 11m. The
results of binding free energy analysis were consistent with the
experimental activity.
Compd.
R1
R2
R3
R4
IC50(μM)
11a
11b
11c
11d
11e
-H
-Cl
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Br
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
93.8 8.6
78.4 12.7
82.7 9.3
-F
-H
-CH3
-OCH3
-C(CH3)3
-Br
-H
126.8 14.9
31.6 5.2
-H
11f
-H
246.9 10.9
172.3 7.7
153.9 11.2
117.2 12.3
93.6 17.2
268.7 10.8
64.1 6.3
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
-H
-H
-CH3
-Cl
-F
-H
-H
-H
-H
-NO2
-H
-H
-H
-CH2CH(CH3)2 390.2 17.2
-CH2CH(CH3)2 216.9 16.8
-CH2CH(CH3)2 143.3 13.9
-Cl
-H
-F
-H
-CH3
-OCH3
-C(CH3)3
-Br
-H
-CH2CH(CH3)2
-CH2CH(CH3)2
178.5 7.8
47.5 5.4
-H
-H
-CH2CH(CH3)2 276.7 18.4
-CH2CH(CH3)2 320.3 16.9
-CH2CH(CH3)2 225.4 11.1
-CH2CH(CH3)2 208.9 15.8
6.9 0.3
11s
11t
11u
Bestatin
-H
-H
-CH3
-Cl
-H
To further understand the difference in bioactivity, the preferred
pharmacophore docking was carried out with the highest activity
and lowest activity compounds (11e, 11m). As shown in Fig. 1, two
compounds have been shown to recognize efficiently the S1 and S1΄
Fig. 1: Diagram (LIGPLOT) of the hydrogen bonds and hydrophobic interactions of the compounds 11e and 11m with active-site residues in Escherichia coli APN (PDB:2DQM)
124
Pharmazie 73 (2018)

ORIGINAL ARTICLES
Fig. 2: Plot of binding free energy contribution per residue of both complexes
2 mol/l HCl solution and extracted with ethyl acetate. The organic phase was washed
with saturated brine and dried over anhydrous MgSO4 and concentrated with a rotary
evaporator to get the colorless oil which was stirred in petroleum ether. The white solid
was filtered as the title compound. Yield: 90.9 %; mp: 80.5–81.3 °C.
Table 2: Binding free energy and their components (kJ/mol) calculat-
ed by MM/PBSA
complex
van der
electrostatic
energy
polar solvation
energy
SASA
energy
Binding
energy
waal energy
3.1.3. N-tert. Butoxycarbonyl-L-leucine (6b)
APN-11e
APN-11m
-177.4
-182.6
-276.1
-85.2
281.6
144.0
-19.0
-20.0
-190.1
-143.9
To a solution of L-leucine (5.0 g, 38.1 mmol) in 76 ml 1 mol/l NaOH and 23 ml
1,4-dioxane was added dropwise a solution of 9.15 g (41.9 mmol) di-tert-butyl dicar-
bonate in 30 ml 1,4-dioxane at 0 °C. The reaction mixture was stirred for 0.5 h at 0 °C,
then was reacted overnight at room temperature keeping pH 8-9. After reaction the
mixture was diluted with 100 ml H2O and extracted with n-hexane. The water phase was
acidified to pH 2-3 with citric acid and extracted with ethyl acetate. The organic phase
was washed with saturated brine and dried over anhydrous MgSO4 and concentrated
under reduced pressure to get the colorless oil as the title compound. Yield: 89.1 %.
2.3. Conclusion
We developed a new series of APN inhibitors of furoic peptidomi-
metic derivatives. Most of the compounds have potent inhibitory
activities toward APN. The compound with methoxy a group at
R4-position was found to be more potent than other target derivatives.
Molecular docking, MD simulation and binding free energy calcu-
lation allow to explain possible binding modes and the difference of
inhibitory activities. These data reveal that ligand coordinating with
the catalytic Zn-ion is very important for inhibitory activities.
3.1.4. (2R)-N-(5-Phenyl-1,3,4-thiadiazol-2-yl)-2-(tertbutoxycarbony-
lamino)-propanamide (7a)
The N,N’-carbonyldiimidazole was added in portions to a solution of compound 6a
(3.60 g, 19 mmol) in 60 ml tetrahydrofuran and stirred for 2 h at room temperature. The
mixture was added to compound 3a (3.81 g, 18 mmol) and stirred for 24 h at 50 °C.
After reaction the solvent was removed under reduced pressure. The residual was
dissolved in 30 ml ethyl acetate and washed with 1 mol/l HCl (3×50ml), H2O (1×50ml),
saturated Na2CO3 (3×50ml) and brine (3×50ml) in turn. The organic phase was dried
over anhydrous MgSO4 and concentrated under reduced pressure and recrystallized
from ethanol to get the title compound as white solid. Yield: 76.1 %; mp: 127–129 °C.
The other compounds (7b-7u) were synthesized by the same method.
3. Experimental
3.1. Chemistry: General procedures
Unless otherwise noted, materials were used without further purification which were
obtained from commercial suppliers. IR was recorded on a FTIR-8400 spectrometer.
ESI-MS was determined on an Aglient-1100 series LC/MSD trap spectrometer.
1HNMR spectra was obtained on a Bruker-400. Melting point was determined on a
electrothermal melting point apparatus and is uncorrected.
3.1.5. (2R)-N-(5-Phenyl-1,3,4-thiadiazol-2-yl)-2-amino-propanamide (8a)
To 3.48 g (10 mmol) of compound 7a was added dropwise a solution of 15 ml trifluoro-
acetic acid in 30 ml dichloromethane. The mixture was reacted for 2 h at room tempera-
ture. After reaction the solvent was removed under reduced pressure. The residual
was dissolved in cooled ethyl acetate, and washed with saturated Na2CO3 to pH 8-9.
The organic phase was dried over anhydrous MgSO4 and concentrated under reduced
pressure to get the title compound as white solid. Yield: 75.0 %; mp: 215–217 °C.
The other compounds (8b-8u) were synthesized by the same method.
3.1.1. 5-Substituted-1,3,4-thiadiazol-2-amines (3a-3l)
The synthetic approach to 5-substituted-1,3,4-thiadiazol-2-amine derivatives was
adapted from the literature (Foronmadi et al. 1999). Briefly, a mixture of carboxylic
acid derivatives (50 mmol), POCl3 (13 ml) and N-aminothiourea (50 mmol) was
reacted for 0.5 h at 75 °C. After cooling down to 0 °C, water was added. The reaction
mixture was refluxed for 4 h. After reaction, the mixture was basified to pH 8 by the
dropwise addition of 50% NaOH solution. The precipitate was filtered and recystal-
lized from ethanol to get the title compounds.
3.1.6. 2-Furoyl chloride (10)
A stirring mixture of 5.6 g (50 mmol) 2-furoic acid and 18 ml SOCl2 was refluxed
for 2 h, and then concentrated under reduced pressure to remove excess SOCl2. The
residual was dissolved in dichloromethane and concentrated under reduced pressure
again to get the title compound as colorless liquid.
3.1.2. N-tert. Butoxycarbonyl-D-alanine (6a)
To a solution of compound D-alanine (6.23 g, 70 mmol) and triethylamine (11.76 ml, 84
mmol) in 40 ml H2O and 20 ml tetrahydrofuran, was added dropwise a solution of 15.28 g
(70 mmol) di-tert-butyl dicarbonate in 20 ml tetrahydrofuran at 0 °C. The reaction mixture
wasstirredfor0.5hat0°C, thenwasreactedovernightatroomtemperaturekeepingpH8-9.
After cooling to 0 °C the mixture was acidified to pH 2-3 by the dropwise addition of
3.1.7. (2R)-N-(5-Phenyl-1,3,4-thiadiazol-2-yl)-2-furoylamino-propana-
mide (11a)
To a solution of compound 8a (1.24 g, 5 mmol) and 4 ml triethylamine in 30 ml dichloro-
methane was added dropwise a solution of 5.5 mmol 2-furoyl chloride in 10 ml
Pharmazie 73 (2018)
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ORIGINAL ARTICLES
dichloromethane at 0 °C. The reaction mixture was stirred for 15 min at 0 °C, then
(2S)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11o): White solid, yield: 61.7%; mp: 193–194 °C. IR (KBr, σ/cm-1):
3130, 2958, 1733, 1637, 1593, 659; 1H NMR (CDCl3) δ 8.65 (d, J = 6.7 Hz, 1H), 8.02
(dd, J=7.7, 5.3Hz, 2H), 7.29–7.20(m, 2H), 7.08–6.98(m, 2H), 6.36(d, J=1.5Hz, 1H),
4.94 (dd, J = 10.2, 6.3 Hz, 1H), 2.06–1.90 (m, 2H), 1.83 (dd, J = 11.4, 6.2 Hz, 1H),
1.00 (dd, J = 10.0, 6.2 Hz, 6H). ESI-MS: m/z [M+H]+ 403.1
(2S)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11p): White solid, yield: 64.9%; mp: 184–186 °C, [α]25 -162.33
(C=0.1 M, CHCl3). IR (KBr, σ/cm-1): 3153, 2956, 1697, 1635, 1593;D1H NMR
(CDCl3) δ 8.89 (d, J = 6.9 Hz, 1H), 7.91 (d, J = 7.7 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H),
7.02 (d, J = 2.6 Hz, 1H), 6.96 (s, 1H), 6.32 (d, J = 1.5 Hz, 1H), 4.98–4.85 (m, 1H),
2.45 (s, 3H), 2.10–1.91 (m, 2H), 1.82 (dd, J = 11.6, 6.6 Hz, 1H), 1.00 (t, J = 6.3 Hz, 6H).
ESI-MS: m/z [M+H]+ 399.1
was reacted overnight at room temperature, and washed with 1 mol/l HCl (2×50ml),
H2O (1×50ml), saturated Na2CO3 (2×50ml) and brine (1×50ml) in turn. The organic
phase was dried over anhydrous MgSO4 and concentrated under reduced pressure and
recrystallized from ethanol to get the target compound as white solid. Yield: 62.0 %;
mp: 220–222 °C. [α]25D +11.98 ° (C = 0.1 M, CHCl3). IR (KBr, σ/cm-1): 3307, 3132,
1660, 1637, 690; 1H NMR (DMSO-d6) δ 12.88 (s, 1H), 8.73 (d, J = 6.6 Hz, 1H),
7.93 (dd, J = 6.4, 3.0 Hz, 2H), 7.87 (d, J = 0.8 Hz, 1H), 7.55–7.50 (m, 3H), 7.21
(d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.67 (m, 1H), 1.45 (d, J = 7.2 Hz,
3H). ESI-MS: m/z [M+H]+ 343. 1. The other final compounds (11b–11u) were
obtained by the same method.
(2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11b): White solid, yield: 65.7%; mp: 199–201 °C. IR (KBr, σ/cm-1): 3178, 1716,
1697, 1608, 1593; 1H NMR (DMSO-d6) δ 12.92 (s, 1H), 8.73 (d, J = 6.6 Hz, 1H),
7.96 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 0.9 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.21
(d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.67 (m, 1H), 1.46 (d, J = 7.2 Hz, 3H).
ESI-MS: m/z [M-H]- 375.1
(2R)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11c): White solid, yield: 62.6%; mp: 176–177 °C. IR (KBr, σ/cm-1): 3153, 1705,
1647, 1595, 661; 1H NMR (DMSO-d6) δ 12.89 (s, 1H), 8.73 (d, J = 6.6 Hz, 1H),
8.07–7.93 (m, 2H), 7.87 (d, J = 0.9 Hz, 1H), 7.37 (dd, J = 12.3, 5.4 Hz, 2H), 7.21
(d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.66 (m, 1H), 1.45 (d, J = 7.2 Hz, 3H).
ESI-MS: m/z [M+H]+ 361.1
(2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11d): White solid, yield: 61.4%; mp: 221–223 °C. IR (KBr, σ/cm-1): 3408, 3132,
1683, 1654, 1635, 669;1H NMR (DMSO-d6) δ 12.83 (s, 1H), 8.71 (d, J = 6.5 Hz, 1H),
7.84 (dd, J = 21.1, 9.0 Hz, 3H), 7.34 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 3.2 Hz, 1H),
6.65 (d, J = 1.5 Hz, 1H), 4.67 (m, 1H), 2.36 (s, 3H), 1.45 (d, J = 7.2 Hz, 3H). ESI-MS:
m/z [M+H]+ 357.1
(2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propan-
amide (11e): White solid, yield: 62.7%; mp: 161–162 °C. IR (KBr, σ/cm-1): 3408,
1697, 1683, 1654, 669; 1H NMR (DMSO-d6) δ 12.78 (s, 1H), 8.70 (d, J = 6.6 Hz, 1H),
7.90–7.85 (m, 3H), 7.21 (dd, J = 3.5, 0.8 Hz, 1H), 7.10–7.06 (m, 2H), 6.65 (dd, J = 3.5,
1.8 Hz, 1H), 4.70–4.64 (m, 1H), 3.82 (s, 3H), 1.45 (d, J = 7.2 Hz, 3H). ESI-MS: m/z
[M+H]+ 373.1
(2R)-N-[5-(4-Tertbutylphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propana-
mide (11f): White solid, yield: 63.1%; mp: 223–225 °C. [α]25D +21.87 ° (C = 0.1 M,
CHCl3). IR (KBr, σ/cm-1): 3282, 3143, 1714, 1647, 682; 1H NMR (DMSO-d6) δ 12.86
(s, 1H), 8.72 (d, J = 6.6 Hz, 1H), 7.87 (dd, J = 11.3, 4.6 Hz, 3H), 7.55 (d, J = 8.5 Hz, 2H),
7.22 (d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.67 (m, 1H), 1.45 (d, J = 7.2 Hz,
3H), 1.31 (s, 9H). ESI-MS: m/z [M+H]+ 399.1
(2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11g): White solid, yield: 67.3%; mp: 218–220 °C. IR (KBr, σ/cm-1): 3417, 3091,
1716, 1697, 1635; 1H NMR (CDCl3) δ 8.03 (d, J = 6.6 Hz, 1H), 7.87 (d, J = 8.5 Hz, 2H),
7.68 (d, J = 8.5 Hz, 2H), 7.20 (s, 1H), 7.12 (d, J = 3.5 Hz, 1H), 6.43 (d, J = 1.7 Hz, 1H),
4.99 (m, 1H), 1.72 (d, J = 7.3 Hz, 3H). ESI-MS: m/z [M-H]- 421.0
(2S)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11q): White solid, yield: 59.2%; mp: 188–190 °C, [α]25 -161.02
D
(C=0.1 M, CHCl3). IR (KBr, σ/cm-1): 3311, 3153, 1701, 1662, 1596; 1H NMR
(CDCl3) δ 8.89 (s, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.05 (s, 1H), 7.02 (d, J = 4.6 Hz, 2H),
6.98 (s, 1H), 6.32 (dd, J = 3.4, 1.7 Hz, 1H), 4.90 (dd, J = 10.1, 6.0 Hz, 1H), 3.90 (s, 3H),
2.07–1.93 (m, 2H), 1.85–1.76 (m, 1H), 1.00 (t, J = 6.9 Hz, 6H). ESI-MS: m/z [M+H]+
415.1
(2S)-N-[5-(4-Tertbutylphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11r): White solid, yield: 59.7%; mp: 240–242 °C, [α]25 -127.90
D
(C=0.1 M, CHCl3). IR (KBr, σ/cm-1): 3298, 3147, 1662, 1593, 680; 1H NMR (CDCl3)
δ 8.81 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.08–6.96 (m, 2H),
6.33 (dd, J = 3.4, 1.7 Hz, 1H), 4.90 (dd, J = 10.3, 6.9 Hz, 1H), 2.01 (d, J = 7.3 Hz, 2H),
1.82 (s, 1H), 1.38 (s, 10H), 1.01 (t, J = 6.0 Hz, 6H). ESI-MS: m/z [M+H]+ 441.2
(2S)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11s): White solid, yield: 62.8%; mp: 138–140 °C. IR (KBr, σ/cm-1):
3134, 2956, 1716, 1697, 1637; 1H NMR (CDCl3) δ 8.31 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H),
7.68 (d, J = 8.5 Hz, 2H), 7.12–7.06 (m, 2H), 6.39 (dd, J = 3.4, 1.6 Hz, 1H), 4.92 (s, 1H),
1.95 (d, J = 7.5 Hz, 2H), 1.85 (d, J = 12.3 Hz, 1H), 1.01 (t, J = 5.6 Hz, 6H). ESI-MS:
m/z [M+H]+ 465.1
(2S)-N-[5-(3-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11t): White solid, yield: 61.5%; mp: 209–210 °C. IR (KBr, σ/cm-1):
3274, 3126, 1749, 1664, 1589, 690; 1H NMR (CDCl3) δ 8.86 (d, J = 6.8 Hz, 1H),7.83
(d, J = 6.1 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.02 (d, J = 2.9 Hz,
1H), 6.98 (s, 1H), 6.33 (dd, J = 3.3, 1.6 Hz, 1H), 4.91 (dd, J = 10.6, 6.8 Hz, 1H), 2.45
(s, 3H), 2.11–1.94 (m, 2H), 1.82 (dd, J = 10.4, 5.5 Hz, 1H), 1.02 (t, J = 5.8 Hz, 6H).
ESI-MS: m/z [M+H]+ 399.1
(2S)-N-[5-(3-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11u): White solid, yield: 58.4%; mp: 196–198 °C. IR (KBr, σ/cm-1):
3178, 3031, 2956, 1716, 1697, 1637, 1595, 682; 1H NMR (DMSO-d6) δ 13.03 (s, 1H),
8.67 (d, J = 7.5 Hz, 1H), 7.99 (t, J = 1.7 Hz, 1H), 7.90–7.86 (m, 2H), 7.61–7.54 (m, 2H),
7.23–7.21 (m, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.79 – 4.71 (m, 1H), 1.83 (ddd, J =
13.4, 10.9, 4.7 Hz, 1H), 1.75–1.68 (m, 1H), 1.59 (ddd, J = 13.5, 9.2, 4.5 Hz, 1H), 0.92
(dd, J = 18.2, 6.6 Hz, 6H). ESI-MS: m/z [M+H]+ 419.0
(2R)-N-[5-(3-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11h): White solid, yield: 64.8%; mp: 193–195 °C. IR (KBr, σ/cm-1): 3313, 3151,
1714, 1631, 686;1H NMR (DMSO-d6) δ 12.86 (s, 1H), 8.73 (d, J = 6.6 Hz, 1H), 7.87
(d, J = 0.9 Hz, 1H), 7.80–7.68 (m, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H),
7.21 (d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.66 (m, 1H), 2.38 (s, 3H),
1.45 (d, J = 7.2 Hz, 3H). ESI-MS: m/z [M+H]+ 357.1
(2R)-N-[5-(3-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11i): White solid, yield: 64.8%; mp: 196–197 °C. IR (KBr, σ/cm-1): 3300, 3145,
1699, 1660, 1659, 686; 1H NMR (DMSO-d6) δ 12.94 (s, 1H), 8.72 (d, J = 6.6 Hz, 1H),
7.99 (t, J = 1.8 Hz, 1H), 7.92– 7.85 (m, 2H), 7.65–7.50 (m, 2H), 7.21 (dd, J = 3.5,
0.7 Hz, 1H), 6.65 (dd, J = 3.5, 1.8 Hz, 1H), 4.68 (m, 1H), 1.46 (d, J = 7.2 Hz, 3H).
ESI-MS: m/z [M+H]+ 377.0
3.2. In-vitro APN inhibition assay
Inhibitory activity toward APN was determined by hydrolysis substrate in 50 mM
PBS (pH 7.2) (Jin et al. 2013). The enzyme is microsomal aminopeptidase from
Porcine Kidney Microsomes and the substrate is L-leu-p-nitroanilide. After adding
the target compounds with various concentrations, the solution was incubated with
APN for 5 min at 37 °C. Then the solution of substrate was added into the above
mixture, which was incubated for another 30 min at 37 oC. Under 405 nm wavelength
the resulting solution was detected to gain absorption.
(2R)-N-[5-(3-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
3.3. Molecular docking
(11j): White solid, yield: 64.8%; mp: 196–197 °C. IR (KBr, σ/cm-1): 3425, 1697, 1636,
1
1593, 682; H NMR (DMSO-d6) δ 12.95 (s, 1H), 8.74 (d, J = 6.5 Hz, 1H), 7.87 (s,
It is necessary to elucidate of ligand binding mechanisms. Molecular docking and
molecular dynamics were studied. The critical amino acid residues were identified for
the most active compound 11e and the least active compound 11m with APN (PDBid:
2DQM). Docking of two compounds to APN was carried out by AutoDock4.2 soft-
ware package with a standard protocol (Morris et al. 1998). All torsion angles for
compounds were considered flexible. In order to include not only the active site but
also significant portions of the surrounding surface, the dimensions of the grids were
set to be sufficiently large for 60×60×60 Å with AutoGrid.
1H), 7.82–7.72 (m, 2H), 7.58 (dd, J = 8.2, 6.1 Hz, 1H), 7.37 (dd, J = 8.4, 1.8 Hz, 1H),
7.21 (d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H), 4.67 (m, 1H), 1.45 (d, J = 7.2 Hz,
3H). ESI-MS: m/z [M+H]+ 361.1
(2R)-N-[5-(2-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-propanamide
(11k): White solid, yield: 66.2%; mp: 204–206 °C. IR (KBr, σ/cm-1): 3365, 3134, 1699,
1697, 1652, 705;1H NMR (DMSO-d6) δ 12.94 (s, 1H), 8.72 (d, J = 6.5 Hz, 1H), 7.93
(dd, J=7.8, 1.6Hz, 1H), 7.88(d, J=0.9Hz, 1H), 7.86–7.82(m, 1H), 7.59–7.52(m, 1H),
7.48 (dd, J = 7.8, 1.7 Hz, 1H), 7.21 (d, J = 3.4 Hz, 1H), 6.65 (dd, J = 3.4, 1.7 Hz, 1H),
4.71–4.64 (m, 1H), 1.46 (d, J = 7.2 Hz, 3H). ESI-MS: m/z [M+H]+ 423.0
(2R)-N-[5-(4-Nitrophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino- propanamide
(11l): Yellow solid, yield: 57.3%; mp: 250–251 °C. IR (KBr, σ/cm-1): 3390, 1716, 1697,
1635, 1524, 1338, 686; 1H NMR (DMSO-d6) δ 13.06 (s, 1H), 8.75 (d, J = 6.6 Hz, 1H),
8.43–8.29 (m, 2H), 8.27 – 8.15 (m, 2H), 7.89 (dd, J = 1.7, 0.8 Hz, 1H), 7.21 (dd, J = 3.5,
0.8 Hz, 1H), 6.66 (dd, J = 3.5, 1.7 Hz, 1H), 4.69 (d, J = 7.2 Hz, 1H), 1.47 (d, J = 7.2 Hz,
3H). ESI-MS: m/z [M-H]- 386.1
(2S)-N-(5-Phenyl-1,3,4-thiadiazol-2-yl)-2-furoylamino-4-methyl-pentanamide
(11m): White solid, yield: 66.2%; mp: 204–206 °C. IR (KBr, σ/cm-1): 3175, 3033,
1733, 1635, 688cm; 1H NMR (DMSO-d6) δ 12.95 (s, 1H), 8.65 (d, J = 7.5 Hz, 1H),
7.94–7.92 (m, 2H), 7.88 (dd, J = 1.7, 0.8 Hz, 1H), 7.53 (dd, J = 5.1, 1.8 Hz, 3H), 7.23
(dd, J = 3.5, 0.7 Hz, 1H), 6.65 (dd, J = 3.5, 1.7 Hz, 1H), 4.77–4.72 (m, 1H), 1.83 (ddd, J =
13.3, 10.9, 4.7 Hz, 1H), 1.76–1.68 (m, 1H), 1.59 (ddd, J = 13.5, 9.1, 4.5 Hz, 1H), 0.92
(dd, J = 18.0, 6.6 Hz, 6H). ESI-MS: m/z [M+H]+ 385.1
3.4. Molecular dynamics simulation
Based on the docking results, MD simulation was carried out with the gromacs 4.6.5
(Van Der Spoel et al. 2005). The APN-11e and APN-11m complex were placed in the
center of an octahedron box, neutralized by adding suitable counterions and solvated by
TIP3P water with amber99SB force field (Hornak et al. 2006). The v-rescale tempera-
ture coupling and parrinello-rahman pressure coupling were used in the NPT ensemble.
The nominal charge of +2 was used for the catalytic Zn-ion which was maintained
in the correct ligation state by distance restraints. The Zn-chelating histidine residues
were protonated at the δ-nitrogen (Manzetti et al. 2003). The complexes were first
energy minimized with the steepest descent method; then a 100 ps position restraining
simulation was carried out to relieve close contacts; finally a 5 ns MD simulation was
performed. Periodic boundary conditions were applied to avoid edge effects.
(2S)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-furoylamino-4-methyl-
pentanamide (11n): White solid, yield: 70.9%; mp: 201–202 °C. IR (KBr, σ/cm-1):
3282, 3143, 1697, 1664, 1591, 680; 1H NMR (CDCl3) δ 8.65–8.50 (m, 1H), 8.06
(s, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.49 (d, J = 15.4, 7.9 Hz, 2H), 7.05 (d, J = 2.2 Hz, 2H),
6.37 (s, 1H), 4.99–4.88 (m, 1H), 2.00 (t, J = 9.2 Hz, 2H), 1.83 (dd, J = 10.3, 5.5 Hz, 1H),
1.10–0.98 (m, 6H). ESI-MS: m/z [M+H]+ 419.1
3.5. Binding free energy calculation
For two complex systems, free energy calculations were performed by g_mmpbsa for
200 snapshots which were extracted from the last 2 ns stable MD trajectory (Kumari et
al. 2014). The van der Waals radius of the catalytic Zn-ion was set to 1.6 (Sakharov et
126
Pharmazie 73 (2018)

ORIGINAL ARTICLES
al. 2005). The free energy of each snapshot was calculated for each molecular species
Ito K, NakajimaY, OnoharaY, Takeo M, Nakashima K, Matsubara F, Ito T,Yoshimoto
T (2006) Crystal structure of aminopeptidase N (proteobacteria alanyl amino-
peptidase) from Escherichia coli and conformational change of methionine 260
involved in substrate recognition. J Biol Chem 281: 33664-33676.
Jin K, Zhang XP, Ma CH, XuYY, YuanYM, Xu WF (2013) Novel indoline-2,3-dione
derivatives as inhibitors of aminopeptidase N (APN). Bioorg Med Chem 21: 2663-
2670.
Kumari R, Kumar R, Open Source Drug Discovery Consortium and Lynn A (2014)
g_mmpbsa—A GROMACS tool for high-throughput MM-PBSA calculations.
J Chem Inf Model 54: 1951-1962.
(complex, ligand and protein), then the binding free energy (ΔGbind) was calculated by
Eq. (1). The molecular mechanics energy (ΔGMM) was calculated by the van der Waals
and electrostatic interactions. The solvation free energy (ΔGsol) was composed of the
nonpolar and the polar contributions. Nonpolar salvation free energy was obtained by
Solvent Accessible Surface Area (SASA) model, whereas polar solvation free energy
could be determined with MM/PBSA method by solving the poisson-boltzmann equa-
tion. TΔS represented the entropy term:
ΔGbind = ΔGMM+ΔGsol-TΔS
(1)
Acknowledgments: The project was supported by the Jiangxi Province Science
Foundation (20171BAB205104) and National Natural Science Foundation of China
(81160383, 81260469).
Lassen KM, Lee J, Joullié MM (2010) Synthetic studies of tamandarin B side chain
analogues. J Org Chem 75: 3027-3036.
Luan Y, Xu W (2007) The structure and main functions of aminopeptidase N. Curr
Med Chem 14: 639-647.
Manzetti S, McCulloch DR, Herington AC, van der Spoel D (2003) Modeling of
enzyme–substrate complexes for the metalloproteases MMP-3, ADAM-9 and
ADAM-10. J Comput Aided Mol Des 17: 551-565.
Conflict of interest: The author(s) confirm that this article content has no conflict of
interest.
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ
(1998) Automated docking using a Lamarckian genetic algorithm and an empirical
binding free energy function. J Comput Chem 19: 1639-1662.
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protein on human dermal fibroblasts. Cell Immunol 162: 42-48.
Repic Lampret B, Kidric J, Kralj B, Vitale L, Pokorny M, Renko M (1999) Lapstatin,
a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits auto-
genous aminopeptidases. Arch Microbiol 171: 397-404.
Rzeski W, Matysiak J, Kandefer-Szerszen M (2007) Anticancer, neuroprotective
activities and computational studies of 2-amino-1,3,4-thiadiazole based compound.
Bioorg Med Chem 15: 3201-3207.
Sakharov DV, Lim C (2005) Zn protein simulations including charge transfer and
local polarization effects. J Am Chem Soc 127: 4921-4929.
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