Novel aminopeptidase N inhibitors derived from 1,3,4-thiadiazole scaffold

Article abstract of DOI:10.1016/j.bmc.2008.05.081

The aminopeptidase N (APN/CD13), overexpressed in tumor cells, plays a critical role in angiogenesis. In this study, we report the synthesis and in vitro enzyme inhibition assay of 1,3,4-thiadiazole scaffold compounds. These new compounds have potent inhi

Full text of DOI:10.1016/j.bmc.2008.05.081

Bioorganic & Medicinal Chemistry 16 (2008) 6663–6668
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel aminopeptidase N inhibitors derived from 1,3,4-thiadiazole scaffold
GuoGang Tu a,b, ShaoHua Li b, HuiMing Huang b, Gang Li b, Fang Xiong c, Xi Mai b, HuaWei Zhu a,
BinHai Kuang b, Wen Fang Xu a,
*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, ShanDong University, 44, West Culture Road, Ji’nan 250012, PR China
b Department of Pharmacy, NanChang University Medical College, 603, BaYi Road, NanChang 330006, PR China
c Department of Cardiology, The Second Affiliated Hospital of NanChang Universit, 1, MinDe Road, NanChang 330006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The aminopeptidase N (APN/CD13), overexpressed in tumor cells, plays a critical role in angiogenesis. In
this study, we report the synthesis and in vitro enzyme inhibition assay of 1,3,4-thiadiazole scaffold com-
pounds. These new compounds have potent inhibitory activities toward APN with IC50 values in the mic-
romol rang.
Received 5 May 2008
Revised 30 May 2008
Accepted 31 May 2008
Available online 5 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Aminopeptidase N
Enzyme inhibitors
Peptidomimetics
1. Introduction
them are currently investigated for clinical uses.18 APN inhibition
by Bestatin leads to a loss of motility. Likewise, CD13 antibodies in-
Aminopeptidase N (APN), also known as CD13, is a widespread
ectopeptidase which preferentially releases neutral and basic ami-
no acids from the N-terminal end of peptides.1 In the intestinal
brush border, APN is involved in the terminal degradation of small
peptides. In synaptic membranes, the enzyme inactivates neuro-
peptides (endorphins and enkephalins).2 APN was also shown to
be the major receptor for the TGEV and HCV229E, and Bacillus thur-
ingensis Cry1A toxin.3–5 Many experimental results suggest that
APN is involved in the down-regulation of several biological active
molecules, such as fMLP, IL-8, angiotensin III, and major histocom-
patibility complex (MHC) class II molecules.6–11 APN plays an
essential role in the entry of HIV into host cells.12 Furthermore, tu-
mor cells which overexpress APN (such as melanoma cells, acute
lymphocytic leukemic cells, and urological cancer cells) are highly
motile and capable of migration through extracellular matrix.13,14
On the other hand, angiogenesis emanating from microvascular
endothelial cells plays a central role in tumor growth and metasta-
sis.15 APN antagonists (CD13 antibodies or bestatin) significantly
block induced-retinal neovascularization in mice and in chorioa-
llantoic membrane angiogenesis in vitro.16,17 All these findings
make this enzyme an interesting target for possible therapeutic
applications, which require the development of potent and selec-
tive inhibitors. To date, several inhibitors of APN, including Besta-
tin, Amastatin, and Actinonin, have been developed and some of
hibit cell growth and cell motility.19–21
Inhibitors containing zinc-binding groups (ZBGs) should be de-
signed to inhibit the activity of APN. This class of inhibitors can
interact with the zinc ions of zinc-dependent metalloenzymes
and sequentially inhibit the metastatic spread of tumors and block
the processes of tumor neovascularization.22 There are two hydro-
phobic domains beside the catalytic activity center of APN, called
pockets S1 and S01, respectively. APN exhibits a broad specificity
for peptides with a N-terminal neutral or basic amino acid such
as alanine, arginine or leucine. Thiadiazole derivatives and cin-
namic acid have been known to have broad biological activities,
especially anti-tumor properties.23,24 According to the ‘combina-
tion principles’, if we link thiadiazole with alanine and cinnamic
acid, the resulting 1,3,4-thiadiazole scaffold compounds should
be capable of inhibiting the enzymatic activity of APN.
2. Chemistry
In order to study the SAR of these novel peptidomimetic com-
pounds, different 1,3,4-thiadiazole derivatives were designed and
synthesized via the route outlined in Scheme 1. The readily avail-
able carboxylic acid 4a–g was condensed with N-aminothiourea
5 in presence of Phosphorus oxychloride, yielding 5-substituted-
1,3,4-thiadiazol-2-amine 6a–g.25 The coupling of
D-alanine 7 with
compound 8a–c led to amide 9a–c.26,27 Finally, the intermediate
6a–g and 9a–c were reacted using DCC to give target compounds
1a–g, 2a–g, and 3a–g.
* Corresponding author. Tel./fax: +86 531 883 82264.
E-mail address: xuwenf@sdu.edu.cn (W.F. Xu).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.081

6664
G. G. Tu et al. / Bioorg. Med. Chem. 16 (2008) 6663–6668
2
1
1
2
2
2
2
2
1
2
1
2
2
3
3
Figure 1. The FlexX docking of compound 2d with active-site in Escherichia coli
APN (PDB:2DQM) by WenFang Xu.
3
Scheme 1. Reagents and conditions: (a) POCl3, 110 °C; (b and c) N-hydroxysucci-
nimide, DCC, rt, by WenFang Xu.
hydrogen bond with Gln-821 and Ala-262, respectively. The
hydrophobic parts of phenyl rings are in contact with nonpolar
surface areas of APN. In addition, all of target compounds, except
compound 3e, show inactivity toward MMP-2 which is a zinc-
dependent endopeptidase associated with the tumorigenic
process.
Although the computed information partially supported our
assumption, the exact binding model of the thiadiazole deriva-
tives with APN should be obtained from further X-ray crystal
studies.
3. Results and discussion
All the inhibition results are summarized in Table 1. The inhi-
bition results showed that most of the target compounds display
excellent potency toward APN with IC50 values lying in micro-
molar level. Comparing 2a–g, we could confirm that hydroxyl
group was positively related with the inhibitory activities. This
could be due to hydroxyl group forming hydrogen bond with
the enzyme. The more active compounds are 2d and 2e, which
have been shown to recognize efficiently the S1 and S01 subsites
of APN. To further understand the interactions of 2d with APN,
the preferred pharmacophore docking studies were carried out
via the FlexX flexible-Dock program. The interaction of 2d with
active site of Escherichia coli APN (PDB:2DQM)28 is shown in Figs.
1 and 2.29 The carbonyl group of alanine can interact with the
zinc ion with the distance of 2.85 Å and form hydrogen bond
with Tyr-383. In addition, the hydroxyl and thiadiazole form
4. Conclusion
In conclusion, we have developed a new series of potent APN
inhibitors. Most of compounds have potent inhibitory activities to-
ward APN and could be used as lead compounds for the develop-
ment of low molecular-weight peptidomimetic APN inhibitors.
Because the critical point in designing selective inhibitors is to
optimize the recognition of APN.
5. Experimental
Table 1
In vitro enzyme assay (APN and MMP-2) results for compounds 1-3 and Bestatin
5.1. Enzyme inhibition assay (in vitro)
N
N
O
H
R2
R1
The target compounds were evaluated for inhibitory activity to-
ward APN and MMP-2. The IC50 values against APN were deter-
mined using L-Leu-p-nitroanilide as substrate and microsomal
aminopeptidase from Porcine Kidney Microsomes (Sigma) as the
enzyme in 50 mM PBS, pH 7.2, at 37 °C.30 MMP assay was per-
formed according to the literature.31 The gelatinase(MMP-2), sub-
stance, and inhibitor were dissolved in sodium borate (pH 8.5,
50 mmol/L) and incubated for 30 min at 37 °C, and then 0.03%
TNBS was added and incubated for another 20 min, the resulting
solution was detected under 450 nm wavelength to gain
absorption.
S
N
N
H
O
a
Compound
R1
R2
IC50 /lM
APN
MMP-2
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
AH
AF
ACl
ABr
ANO2
ACH3
AOCH3
AH
AF
ACl
-H
215.6 12.4
183.4 8.7
152.6 11.6
114.9 13.7
84.7 9.0
nab
na
na
na
AH
AH
AH
AH
AH
AH
AOH
AOH
AOH
AOH
AOH
AOH
AOH
AOCH3
AOCH3
AOCH3
AOCH3
AOCH3
AOCH3
AOCH3
na
206.2 15.7
31.4 18.0
123.9 9.7
115.4 12.6
104.9 17.9
35.3 0.9
na
na
na
na
na
na
5.2. Chemistry: general procedures
ABr
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. All the sol-
vents except DMF have been distilled before use. All reactions
were monitored by thin-layer chromatography on 0.25 mm silica
gel plates (60GF-254) and visualized with UV light. Column chro-
matography was performed on silica gel (200–300 mesh). ESI-MS
was determined on a Aglient-1100 series LC/MSD trap spectrome-
ter. IR was recorded on a FTIR-8400 spectrometer. Melting point
was determined on a electrothermal melting point apparatus and
is uncorrected. 1H NMR spectra was obtained on a brucker-400.
The chemical shifts are expressed in d values (parts per million)
relative to tetramethylsilane (TMS) as internal standard. Signifi-
ANO2
ACH3
AOCH3
AH
AF
ACl
54.1 8.4
na
126.8 12.9
68.8 7.9
na
na
131.1 14.4
90.7 20.8
134.6 15.2
108.2 11.9
44.6 10.7
141.1 12.8
78.5 0.6
na
na
na
na
ABr
ANO2
ACH3
AOCH3
Bestatin
297.3 28.5
na
na
3g
8.5 0.6
9.5 0.5
a
Values are means of three experiments, standard deviation is given.
not activity.
b

G. G. Tu et al. / Bioorg. Med. Chem. 16 (2008) 6663–6668
6665
Figure 2. Diagram (LIGPLOT) of the hydrogen bonds and hydrophobic interactions of the compound 2d with active-site residues in Escherichia coli APN (PDB:2DQM) by
WenFang Xu.
cant 1H NMR data are reported in the following order: multiplicity
(s, singlet; d, doublet; t, triplet; m, multiplet) number of protons.
J1 = 6.38 Hz, J2 = 14.00 Hz, 2H), 7.74–7.76 (d, J = 6.94 Hz, 2H), ESI-
MS: m/z [M+H]+ 178.3.
The following compounds were prepared according to the gen-
eral procedure as described for the preparation of compound 6a
starting from compounds 4a–g by coupling with compound 5.
5.2.1. General procedure for preparing 5-substituted-1,3,
4-thiadiazol-2-amine. Synthesis of 5-phenyl-1,3,4-thiadiazol-2-
amino (6a)
A stirring mixture of benzoic acid (50 mmol), N-aminothiourea
(50 mmol) and POCl3 (13 ml) was heated at 75 °C for 0.5 h. After
cooling down to room temperature, water was added. The reaction
mixture was refluxed for 4 h. After cooling, the mixture was basi-
fied to pH 8 by the dropwise addition of 50% NaOH solution under
stirring. The precipitate was filtered and recystallized from ethanol
to yield 7.0 g (79.1%) of the title compounds as white solid; mp:
5.2.2. 5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-amino (6b)
White solid, yield: 82.5%; mp: 232–234 °C; IR (KBr,
3341, 3246 ( NH), 1061 ( CAF), 687(
CAS); 1H NMR (DMSO-d6): d
r
/cmꢀ1):
t
t
t
7.29–7.33 (t, J = 8.77 Hz, 2H), 7.41 (s, 2H), 7.79–7.83 (dd,
J1 = 5.46 Hz, J2 = 8.59 Hz, 2H), ESI-MS: m/z [M+H]+ 196.4.
5.2.3. 5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-amino (6c)
224–226 °C; IR (KBr,
r
/cmꢀ1): 3276, 3114 (
7.40 (s, 2H), 7.43–7.49 (dd,
t
NH), 1634 (tC@N), 691
White solid, yield: 77.4%; mp: 214–216 °C; IR (KBr,
3269, 3098 ( NH), 1636 ( C@N), 694 (
CAS); 1H NMR (DMSO-d6): d
r
/cmꢀ1):
(t
CAS); 1H NMR (DMSO-d6):
d
t
t
t

6666
G. G. Tu et al. / Bioorg. Med. Chem. 16 (2008) 6663–6668
7.47 (s, 2H), 7.52–7.54 (d, J = 6.55 Hz, 2H), 7.76–7.78 (d, J = 6.24 Hz,
2H), ESI-MS: m/z [M+H]+ 212.6.
J = 15.76 Hz, 1H), 6.97–7.00 (d, J = 8.72 Hz, 2H), 7.39–7.43 (d,
J = 15.80 Hz, 1H), 7.51–7.54 (d, J = 8.76 Hz, 2H), 8.31–8.33 (d,
J = 7.40 Hz, 1H), 12.57 (s, 1H). ESI-MS: m/z [M+H]+ 250.2.
5.2.4. 5-(4-Bromophenyl)-1,3,4-thiadiazol-2-amino (6d)
White solid, yield: 84.1%; mp: 222–224 °C; IR (KBr,
r
/
5.2.11. General procedure for preparing propanamide.
Synthesis of (2R)-N-[5-phenyl-1,3,4-thiadiazol-2-yl]-2-
[(cinnamoyl)amino]-propanamide (1a)
cmꢀ1):3286, 3090 CAS); 1H NMR
(
t
NH), 1638 C@N), 690
(
t
(t
(DMSO-d6):
d
7.47 (s, 2H), 7.65–7.72 (dd, J1 = 8.48 Hz,
J2 = 16.97 Hz, 4H), ESI-MS: m/z [M+H]+ 256.2.
N,N-Dicyclohexylcarbodiimide (5.7 mmol) was added to
a
cooled solution of N-cinnamoyl- -alanine (9a) (5.6 mmol) and N-
D
5.2.5. 5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-amino (6e)
hydroxysuccinimide (5.6 mmol) in freshly distillation dioxane
(30 ml). The reaction mixture was stirred overnight at room tem-
perature. The insoluble material was filtered off and washed with
cold dioxane. 5-Phenyl-1,3,4- thiadiazol-2-amino (6a) (5.5 mmol)
was added to the filtrate and the reaction mixture was stirred for
48 h at room temperature. The solvent was removed under re-
duced pressure. The residual was dissolved in EtOAc and the insol-
uble material was filtered off. The filtrate was washed successively
with saturated Na2CO3 solution (3ꢁ 20 ml), water (1ꢁ 20 ml),
0.1 M HCl (3ꢁ 20 ml) and water (1ꢁ 20 ml). The organic layer
evaporated in vacuo, the residual was recrystallized from metha-
nol/acetonitrile (5:1) and dried to afford the target compound as
Yellow solid, yield: 80.6%; mp: 250–252 °C; IR (KBr,
r
/cmꢀ1):
3398, 3288 (
t
NH), 1630 (
tC@N), 691 (
t
CAS), 1506 (tNO ); 1H NMR
2
(DMSO-d6): d 7.74 (s, 2H), 8.00–8.01 (d, J = 2.20 Hz, 2H), 8.28–
8.29 (d, J = 2.28 Hz, 2H); ESI-MS: m/z [M+H]+ 223.3.
5.2.6. 5-(4-Methylphenyl)-1,3,4-thiadiazol-2-amino (6f)
White solid, yield: 90.2%; mp: 210–211 °C; IR (KBr,
r/
cmꢀ1):3288, 3100 ( CH); 1H
t
NH), 1635 ( C@N), 690 ( CAS), 2952 (
t
t
t
NMR (DMSO-d6): d 2.33(s, 3H), 7.25–7.27 (d, J = 8.00 Hz, 2H),
7.36 (s, 2H), 7.63–7.65 (d, J = 8.12 Hz, 2H); ESI-MS: m/z [M+H]+
192.2.
white solid 0.34 g, yield: 16.3%. mp: 209–210 °C. IR (KBr,
r/
5.2.7. 5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-amino (6g)
cmꢀ1): 3417, 3262 (
tNH), 1684,1654 (tC@O), 690 (tCAS), 1620 (tC@N).
White solid, yield: 92.3%; mp: 192–194 °C; IR (KBr,
/cmꢀ1):
3286, 3107 ( NH), 1628 ( C@N), 1254 ( ArAOAC), 2960 (t
CH); 1H
r
1H NMR (DMSO-d6) d: 1.42–1.43 (d, J = 7.16 Hz, 3H), 4.64–4.71(m,
1H), 6.74–6.78 (d, J = 15.96 Hz, 1H), 7.40–7.48 (m, 4H), 7.53–7.60
(m, 5H), 7.94–7.96 (m, 2H), 8.61–8.63 (d, J = 6.48 Hz, 1H), 12.86
(s, 1H). ESI-MS: m/z [M+H]+ 379.2.
t
t
t
NMR (DMSO-d6): d 3.80 (s, 2H), 7.02 (s, 2H), 7.27 (s, 2H), 7.68 (s,
2H), ESI-MS: m/z [M+H]+ 208.2.
The following compounds were prepared according to the gen-
eral procedure as described for the preparation of compound 1a
starting from compounds 9a–c by coupling with compounds 6a–g.
5.2.8. General procedure for preparing N-acyl-
Synthesis of N-cinnamoyl- -alanine (9a)
N,N-Dicyclohexylcarbodiimide (32 mmol) was added to
D-alanine.
D
a
cooled solution of cinnamic acid (30 mmol) and N-hydroxy-
succinimide (32 mmol) in freshly distillation dioxane (46 ml). The
reaction mixture was stirred for 5 h at room temperature. The
insoluble material was filtered off and washed with cold dioxane.
5.2.12. (2R)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-
[(cinnamoyl)amino]-propanamide (1b)
White solid, yield: 28.3%; mp: 249–250 °C; IR (KBr,
/cmꢀ1):
3402, 3261 ( NH), 1701, 1658 ( C@O), 686 ( CAS), 1623 (t
C@N); 1H
r
t
t
t
The filtrate was added to a solution of
D
-alanine (34 mmol) and
NMR (DMSO-d6): d 1.40–1.42 (d, J = 7.08 Hz, 3H), 4.63–4.66 (t,
J = 6.86 Hz, 1H), 6.72-6.76 (d, J = 15.88 Hz, 1H), 7.36–7.47 (m, 6
H), 7.57–7.59 (d, J = 7.24 Hz, 2H), 7.99–8.02 (dd, J1 = 5.56 Hz,
J2 = 8.24 Hz, 2H), 8.64–8.66 (d, J = 6.40 Hz, 1H), 12.90 (s, 1H); ESI-
MS: m/z [M+H]+ 397.1.
NaHCO3 (34 mmol) in H2O (48 ml) and the reaction mixture stirred
at room temperature for 24 h. The solvent was removed under re-
duced pressure. The residual was dissolved in water and the insol-
uble material was filtered off. The aqueous layer was extracted
with EtOAc (3ꢁ 30 ml). The resulting aqueous layer was acidified
with concentrated HCl (pH 2) and allowed to cool. The precipitate
formed was filtered, recrystallized from methanol and dried to af-
ford the target compound as white solid 3.7 g, yield: 56.6%. mp:
5.2.13. (2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-
[(cinnamoyl)amino]- propanamide (1c)
White solid, yield: 39.4%; mp: 207–208 °C; IR (KBr,
/cmꢀ1):
3437, 3155 ( NH), 1701, 1659 ( C@O), 686 ( CAS), 1620 (t
C@N); 1H
r
154–156 °C; IR (KBr,
r
/cmꢀ1): 3323 (
t
NH), 1656 (
t
C@O), 2943
t
t
t
(t
OH), 1724 (
t
C@O). 1H NMR (DMSO-d6): d 1.35–1.37 (d, J =
NMR (DMSO-d6): d 1.41–1.43 (d, J = 7.20 Hz, 3H), 4.64–4.71 (m,
1H), 6.73–6.77 (d, J = 15.92 Hz, 1H), 7.39–7.48 (m, 4H), 7.58–7.61
(m, 4H), 7.96–7.98 (dd, J1 = 1.92 Hz, J2 = 6.60 Hz, 2H), 8.62–8.63
(d, J = 6.48 Hz, 1H), 12.91 (s, 1H); ESI-MS: m/z [M+H]+ 413.1.
7.28 Hz, 3H), 4.35–4.42(m, 1H), 6.73–6.77 (d, J = 15.84 Hz, 1H),
7.37–7.50 (m, 4H), 7.58–7.59 (d, J = 7.28 Hz, 2H), 8.42–8.44 (d,
J = 7.32 Hz, 1H), 12.62 (s, 1H). ESI-MS: m/z [M+H]+ 220.2.
5.2.9. N-(p-Coumaroyl)-
D
-alanine (9b)
5.2.14. (2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-
The title compound was prepared starting from p-coumaric acid
as described for compound 9a. Yield: 62.6%; white solid; mp: 188–
[(cinnamoyl)amino]-propanamide (1d)
White solid, yield: 14.9%; mp: 158–60 °C; IR (KBr,
/cmꢀ1):
3301, 3166 ( NH), 1698, 1659 ( C@O), 688 ( CAS), 1622 (t
C@N); 1H
r
190 °C. IR (KBr,
r
/cmꢀ1): 3325 (
t
NH), 1647 (
t
C@O), 3099 (
t
OH), 1724
t
t
t
(t
C@O). 1H NMR (DMSO-d6): d 1.33–1.34 (d, J = 7.28 Hz, 3H), 4.31–
NMR (DMSO-d6): d 1.40–1.42 (d, J = 7.12 Hz, 3H), 4.64–4.67 (t,
J = 6.86 Hz, 1H), 6.72–6.76 (d, J = 15.80 Hz, 1H), 7.47–7.51 (d,
J = 16.64 Hz, 1H), 7.58–7.72 (m, 5H), 7.73–7.75 (d, J = 8.48 Hz,
2H), 7.89–7.91 (d, J = 8.48 Hz, 2H), 8.66–8.67 (d, J = 6.40 Hz, 1H),
12.95 (s, 1H); ESI-MS: m/z [M+H]+458.0.
4.39(m, 1H), 6.49–6.53 (d, J = 15.76 Hz, 1H), 6.81–6.83 (d,
J = 8.55 Hz, 2H), 7.34–7.43 (m, 3H), 8.26–8.28 (d, J = 7.36 Hz, 1H),
9.62–9.64 (d, J = 7.45 Hz, 1H), 12.59 (s, 1H). ESI-MS: m/z [M+H]+
236.3.
5.2.10. N-(p-Methoxycinnamoyl)-D-alanine (9c)
5.2.15. (2R)-N-[5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-yl]-2-
The title compound was prepared starting from p-methoxycin-
namic acid as described for compound 9a. Yield: 68.2%; white so-
[(cinnamoyl)amino]-propanamide (1e)
Yellow solid, yield: 50.2%; mp: 265–267 °C; IR (KBr,
r
/cmꢀ1):
tNH), 1700, 1659 (tC@O), 688 (tCAS), 1617 (tC@N),
lid; mp: 162–164 °C. IR (KBr,
3018 ( OH), 1717 (
C@O). 1H NMR (DMSO-d6): d 1.33–1.35 (d,
J = 7.28 Hz, 3H), 3.79 (s, 3H), 4.33–4.40 (m, 1H), 6.57–6.1 (d,
r
/cmꢀ1): 3375 (
t
NH), 1650 (
t
C@O),
3422, 3320 (
t
t
1522 ( asNO ), 1333 (
t
t
sNO ); 1H NMR (DMSO-d6): d 1.39–1.41 (d,
2
2
J = 6.86 Hz, 3H), 4.59–4.63 (t, J = 6.78 Hz, 1H), 6.78–6.82 (d,

G. G. Tu et al. / Bioorg. Med. Chem. 16 (2008) 6663–6668
6667
J = 15.82 Hz, 1H), 7.39–7.46 (dd, J1 = 10.37 Hz, J2 = 16.32 Hz, 4H),
7.58–7.60 (d, J = 7.12 Hz, 2H), 8.18–8.20 (d, J = 8.30 Hz, 2H), 8.33–
8.35 (d, J = 8.41 Hz, 2H), 8.48 (s, 1H), 12.96 (s, 1H); ESI-MS: m/z
[M+H]+ 424.1.
5.2.22. (2R)-N-[5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-yl]-2-
[(p-coumaroyl)amino]-propanamide (2e)
Yellow solid, yield: 23.1%; mp: 225–227 °C; IR (KBr,
r
/cmꢀ1):
C@N), 1517
1.40–1.42 (d,
3429, 3327 (
tNH), 1696, 1654 (tC@O), 690 (tCAS), 1621 (t
(t
asNO ), 1345 (
t
sNO ); 1H NMR (DMSO-d6):
d
2
2
5.2.16. (2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-
J = 7.12 Hz, 3H), 4.63–4.66 (t, J = 6.88 Hz, 1H), 6.50–6.53 (d,
J = 15.80 Hz, 1H), 6.80–6.82 (d, J = 8.44 Hz, 2H), 7.33–7.37 (d,
J = 15.76 Hz, 1H), 7.40–7.42 (d, J = 8.44 Hz, 2H), 8.23–8.25 (d,
J = 8.76 Hz, 2H), 8.35–8.38 (d, J = 8.79 Hz, 2H), 8.50–8.51 (d,
J = 6.18 Hz, 1H), 9.89 (s, 1H), 13.04(s, 1H);ESI-MS: m/z [M+H]+ 440.2.
[(cinnamoyl)amino]-propanamide (1f)
White solid, yield: 29.3%; mp: 210–212 °C; IR (KBr,
/cmꢀ1):
3427, 3310 ( NH), 1703, 1660 ( C@O), 691 ( CAS), 1621 (t
C@N); 1H
r
t
t
t
NMR (CDCl3): d 1.66–1.68 (d, J = 7.40 Hz, 3H), 2.42 (s, 3H), 4.92–
5.00 (m, 1H), 6.39–6.43 (d, J = 15.72 Hz, 1H), 7.05–7.26 (m, 4H),
7.35–7.38 (t, J = 6.32 Hz, 2H), 7.53–7.56 (d, J = 15.72 Hz, 1H),
7.86–7.88 (d, J = 8.08 Hz, 2H), 8.65–8.67 (d, J = 7.04 Hz, 1H), 13.33
(s, 1H); ESI-MS: m/z [M+H]+ 393.3.
5.2.23. (2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-[(p-
coumaroyl)amino]-propanamide (2f)
White solid, yield: 35.8%; mp: 195–197 °C; IR (KBr,
3333, 3253 ( NH), 1710, 1655 ( C@O), 690 (
CAS); 1H NMR (DMSO-
r
/cmꢀ1):
t
t
t
5.2.17. (2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-
d6): d 1.39–1.41 (d, J = 7.08 Hz, 3H), 2.37 (s, 3H), 4.62–4.65 (t,
J = 6.88 Hz, 1H), 6.51–6.55 (d, J = 15.76 Hz, 1H), 6.80–6.82 (d,
J = 8.40 Hz, 2H), 7.33–7.37 (t, J = 7.70 Hz, 3H), 7.41–7.43 (d,
J = 8.44 Hz, 2H), 7.82–7.84 (d, J = 7.96 Hz, 2H), 8.50–8.52 (d,
J = 6.36 Hz, 1H), 9.93(s, 1H), 12.84(s, 1H); ESI-MS:m/z[M+H]+ 409.1.
[(cinnamoyl)amino]-propanamide (1g)
White solid, yield: 35.2%; mp: 176–178 °C; IR (KBr,
3437, 3209 ( NH), 1670, 1654 ( C@O), 1627 (
C@N); 1H NMR
r
/cmꢀ1):
t
t
t
(DMSO-d6): d 1.40–1.42 (d, J = 7.12 Hz, 3H), 3.83 (s, 3H), 4.64–
4.67 (t, J = 6.90 Hz, 1H), 6.72–6.76 (d, J = 15.88 Hz, 1H), 7.08–7.10
(d, J = 8.72 Hz, 2H), 7.39–7.47 (m, 4H), 7.57–7.59 (d, J = 7.08 Hz,
2H), 7.87–7.89 (d, J = 8.72 Hz, 2H), 8.59–8.61 (d, J = 6.36 Hz, 1H),
12.77 (s, 1H); ESI-MS: m/z [M+H]+ 409.1.
5.2.24. (2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-
[(p-coumaroyl)amino]-propanamide (2g)
White solid, yield: 42.1%; mp: 215–216 °C; IR (KBr,
3408, 3272 ( NH), 1695, 1655 (
C@O); 1H NMR (DMSO-d6): d 1.38–
r
/cmꢀ1):
t
t
5.2.18. (2R)-N-[5-Phenyl-1,3,4-thiadiazol-2-yl]-2-
[(p-coumaroyl)amino]-propanamide (2a)
1.40 (d, J = 7.16 Hz, 3H), 3.83 (s, 3H), 4.62 (s, 1H), 6.50–6.54 (d,
J = 15.92 Hz, 1H), 6.79–6.81 (d, J = 8.48 Hz, 2H), 7.07–7.10 (d,
J = 8.72 Hz, 2H), 7.32–7.36 (d, J = 15.76 Hz, 1H), 7.40–7.42 (d,
J = 8.52 Hz, 2H), 7.86–7.89 (d, J = 8.68 Hz, 2H), 8.47 (s, 1H), 9.88
(s, 1H), 12.75 (s, 1H); ESI-MS: m/z [M+H]+ 425.1
White solid, yield: 24.9%; mp: 215-217 °C; IR (KBr,
/cmꢀ1):
3389, 3327 ( NH), 1700, 1651 ( C@O), 690 ( CAS), 1628 (t
C@N); 1H
r
t
t
t
NMR (DMSO-d6): d 1.39–1.41 (d, J = 7.04 Hz, 3H), 4.64 (s, 1H),
6.50–6.54 (d, J = 15.64 Hz, 1H), 6.80–6.82 (d, J = 8.48 Hz, 2H),
7.33–7.37 (d, J = 15.78 Hz, 1H), 7.40–7.42 (d, J = 8.46 Hz, 2H),
7.53–7.54 (d, J = 2.08 Hz, 3H), 7.93–7.94 (d, J = 3.40 Hz, 2H), 8.46–
8.48 (d, J = 6.24 Hz, 1H), 9.87 (s, 1H), 12.83 (s, 1H); ESI-MS: m/z
[M+H]+ 395.0.
5.2.25. (2R)-N-[5-Phenyl-1,3,4-thiadiazol-2-yl]-2-[(p-
methoxycinnamoyl)amino]-propanamide (3a)
White solid, yield: 23.5%; mp: 145–147 °C; IR (KBr,
/cmꢀ1):
3405, 3286 ( NH), 1700, 1650 ( C@O), 691 ( CAS), 1620 (t
C@N); 1H
r
t
t
t
NMR (DMSO-d6): d 1.39–1.41 (d, J = 6.96 Hz, 3H), 3.79 (s, 3H),
4.63–4.65 (d, J = 6.92 Hz, 1H), 6.57–6.61 (d, J = 15.72 Hz, 1H),
6.98–7.00 (d, J = 8.16 Hz, 2H), 7.37–7.41 (d, J = 16.32 Hz, 1H), 7.54
(s, 5H), 7.93–7.94 (d, J = 4.48 Hz, 2H), 8.85–8.55 (d, J = 6.08 Hz,
1H), 12.88 (s, 1H); ESI-MS: m/z [M+H]+ 409.1.
5.2.19. (2R)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-
[(p-coumaroyl)amino]-propanamide (2b)
White solid, yield: 23.3%; mp: 220–223 °C; IR (KBr,
3262, 3159 ( NH), 1695, 1655 (
C@O); 1H NMR (DMSO-d6): d 1.38–
r
/cmꢀ1):
t
t
1.40 (d, J = 7.12 Hz, 3H), 4.61–4.64 (t, J = 6.86 Hz, 1H), 6.50–6.54
(d, J = 15.76 Hz, 1H), 6.79–6.82 (d, J = 8.48 Hz, 2H), 7.32–7.42 (m,
5H), 7.99–8.02 (dd, J1 = 5.40 Hz, J2 = 8.64 Hz, 2H), 8.50–8.52 (d,
J = 6.36 Hz, 1H), 9.92 (s, 1H), 12.88 (s, 1H); ESI-MS: m/z [M+H]+
413.2.
5.2.26. (2R)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(p-
methoxycinnamoyl)amino]-propanamide (3b)
White solid, yield: 68.4%; mp: 183–185 °C; IR (KBr,
3405,3277 ( NH), 1691,1653 ( C@O), 1621 (
C@N); 1H NMR (DMSO-
r
/cmꢀ1):
t
t
t
d6): d 1.39–1.41 (d, J = 7.12 Hz, 3H), 3.79 (s, 3H), 4.62–4.65 (t,
J = 6.86 Hz, 1H), 6.57–6.61 (d, J = 15.84 Hz, 1H), 6.98–7.01 (d,
J = 8.64 Hz, 2H), 7.36–7.41 (dd, J1 = 9.36 Hz, J2 = 14.84 Hz, 3H),
7.52–7.54 (d, J = 8.64 Hz, 2H), 7.99–8.02 (dd, J1 = 5.40 Hz,
J2 = 8.64 Hz, 2H), 8.54–8.86 (d, J = 6.40 Hz, 1H), 12.89 (s, 1H); ESI-
MS: m/z [M+H]+ 427.1.
5.2.20. (2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-
[(p-coumaroyl)amino]-propanamide (2c)
White solid, yield: 42.8%; mp: 265–266 °C; IR (KBr,
3388, 3165 ( NH), 1685, 1655 ( C@O), 697 (
CAS); 1H NMR (DMSO-
r
/cmꢀ1):
t
t
t
d6): d 1.40–1.41 (d, J = 7.16 Hz, 3H), 4.62–4.68 (m, 1H), 6.51–6.54
(d, J = 15.80 Hz, 1H), 6.80–6.82 (d, J = 8.48 Hz, 2H), 7.34–7.38 (d,
J = 15.76 Hz, 1H), 7.40–7.67 (m, 4H), 7.96–7.98 (t, J = 4.30 Hz, 2H),
8.47–8.28 (d, J = 6.48 Hz, 1H), 9.87 (s, 1H), 12.88 (s, 1H); ESI-MS:
m/z [M+H]+ 429.1.
5.2.27. (2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(p-
methoxycinnamoyl)amino]-propanamide (3c)
White solid, yield: 35.8%; mp: 125–128 °C; IR (KBr,
3405, 3333 ( NH), 1700, 1655 ( C@O), 1627 (
C@N); 1H NMR
r
/cmꢀ1):
t
t
t
5.2.21. (2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-
[(p-coumaroyl)amino]-propanamide (2d)
(DMSO-d6): d 1.39–1.41 (d, J = 7.12 Hz, 3H), 3.79 (s, 3H), 4.62–
4.66 (t, J = 6.84 Hz, 1H), 6.57–6.61 (d, J = 15.80 Hz, 1H), 6.98–7.01
(d, J = 8.52 Hz, 2H), 7.37–7.42 (d, J = 15.76 Hz, 1H), 7.52–7.62 (m,
4H), 7.96–7.98 (d, J = 8.40 Hz, 2H), 8.55–8.57 (d, J = 6.28 Hz, 1H),
12.93 (s, 1H); ESI-MS: m/z [M+H]+ 443.2.
White solid, yield: 32.7%; mp: 242–244 °C; IR (KBr,
3341, 3203 ( NH), 1706, 1653 (
C@O); 1H NMR (DMSO-d6): d 1.38–
r
/cmꢀ1):
t
t
1.40 (d, J = 7.08 Hz, 3H), 4.61–4.64 (t, J = 6.86 Hz, 1H), 6.49–6.53
(d, J = 15.76 Hz, 1H), 6.79–6.81 (d, J = 8.40 Hz, 2H), 7.32–7.36
(d, J = 15.76 Hz, 1H), 7.40–7.42 (d, J = 8.36 Hz, 2H), 7.73–7.75 (d,
J = 8.44 Hz, 2H), 7.89–7.91 (d, J = 8.40 Hz, 2H), 8.50–8.52 (d,
J = 6.28 Hz, 1H), 9.92 (s, 1H), 12.92 (s, 1H); ESI-MS: m/z
[M+H]+474.3.
5.2.28. (2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-[(p-
methoxycinnamoyl)amino]-propanamide (3d)
White solid, yield: 45.2%; mp: 134–135 °C; IR (KBr,
3405, 3285 ( NH), 1698, 1652 ( C@O), 1620 (
C@N); 1H NMR
r
/cmꢀ1):
t
t
t

6668
G. G. Tu et al. / Bioorg. Med. Chem. 16 (2008) 6663–6668
(DMSO-d6): d 1.39–1.41 (d, J = 7.12 Hz, 3H), 3.79 (s, 3H), 4.62–4.65
(t, J = 6.88 Hz, 1H), 6.57–6.61 (d, J = 15.80 Hz, 1H), 6.98–7.00 (d,
J = 8.64 Hz, 2H), 7.37–7.41 (d, J = 15.76 Hz, 1H), 7.52–7.54 (d,
J = 8.64 Hz, 2H), 7.73–7.75 (d, J = 8.48 Hz, 2H), 7.89–7.91 (d,
J = 8.48 Hz, 2H), 8.54–8.56 (d, J = 6.40 Hz, 1H), 12.93 (s, 1H); ESI-
MS: m/z [M+H]+ 488.1.
dation of Ministry of Education of PR China (No. 20060422029) and
Shandong Nature Science Foundation of China (Grant No.
Y2004C02).
References and notes
1. Riemann, D.; Kehlen, A.; Langner, J. Immunol. Today 1999, 20, 83.
2. Hooper, N. M. FEBS Lett. 1994, 354, 1.
3. Delmas, B.; Gelfi, J.; L’Haridon, R.; Vogel, L. K.; Sjostrom, H.; Noren, O.; Laude, H.
Nature 1992, 357, 417.
4. Nakanishi, K.; Yaoi, K.; Nagino, Y.; Hara, H.; Kitami, M.; Atsumi, S.; Miura, N.;
Sato, R. FEBS Lett. 2002, 519, 215.
5. Yeager, C. L.; Ashmun, R. A.; Williams, R. K.; Cardellichio, C. B.; Shapiro, L. H.;
Look, A. T.; Holmes, K. V. Nature 1992, 357, 420.
6. Hattori, A.; Tsujimoto, M. Biol. Pharm. Bull. 2004, 27, 777.
7. Irazusta, J.; Valdivia, A.; Fernandez, D.; Agirregoitia, E.; Ochoa, C.; Casis, L. J.
Androl. 2004, 25, 733.
8. Kanayama, N.; Kajiwara, Y.; Goto, J.; el Maradny, E.; Maehara, K.; Andou, K.;
Terao, T. J. Leukocyte Biol. 1995, 57, 129.
9. Larsen, S. L.; Pedersen, L. O.; Buus, S.; Stryhn, A. J. Exp. Med. 1996, 184, 183.
10. Mitsui, T.; Nomura, S.; Itakura, A.; Mizutani, S. Biol. Pharm. Bull. 2004, 27, 768.
11. Shipp, M. A.; Look, A. T. Blood 1993, 82, 1052.
12. Pulido-Cejudo, G.; Conway, B.; Proulx, P.; Brown, R.; Izaguirre, C. A. Antiviral
Res. 1997, 36, 167.
13. Fujii, H.; Nakajima, M.; Saiki, I.; Yoneda, J.; Azuma, I.; Tsuruo, T. Clin. Exp.
Metastasis 1995, 13, 337.
14. Ishii, K.; Usui, S.; Sugimura, Y.; Yoshida, S.; Hioki, T.; Tatematsu, M.; Yamamoto,
H.; Hirano, K. Int. J. Cancer 2001, 92, 49.
15. Tomanek, R. J.; Schatteman, G. C. Anat. Rec. 2000, 261, 126.
16. Bhagwat, S. V.; Lahdenranta, J.; Giordano, R.; Arap, W.; Pasqualini, R.; Shapiro,
L. H. Blood 2001, 97, 652.
17. Hashida, H.; Takabayashi, A.; Kanai, M.; Adachi, M.; Kondo, K.; Kohno, N.;
Yamaoka, Y.; Miyake, M. Gastroenterology 2002, 122, 376.
18. Sato, Y. Biol. Pharm. Bull. 2004, 27, 5.
19. Antczak, C.; De Meester, I.; Bauvois, B. Bioessays 2001, 23, 251.
20. Lohn, M.; Mueller, C.; Langner, J. Leuk. Lymphoma 2002, 43, 407.
21. Saiki, I.; Fujii, H.; Yoneda, J.; Abe, F.; Nakajima, M.; Tsuruo, T.; Azuma, I. Int. J.
Cancer 1993, 54, 137.
22. Becker, J. W.; Marcy, A. I.; Rokosz, L. L.; Axel, M. G.; Burbaum, J. J.; Fitzgerald, P.
M. D.; Cameron, P. M.; Esser, C. K.; Hagmann, W. K.; Hermes, J. D.; Springer, J. P.
Protein Sci. 1995, 4, 1966.
5.2.29. (2R)-N-[5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(p-
methoxycinnamoyl)amino]-propanamide (3e)
Yellow solid, yield: 51.3%; mp: 265–266 °C; IR (KBr,
r
/cmꢀ1):
3425, 3281 (tNH), 1694, 1655 (tC@O), 690 (tCAS), 1517 (tasNO ),
2
1345 (
t
sNO ); 1H NMR (DMSO-d6): d 1.40–1.42 (d, J = 7.08 Hz,
2
3H), 3.80 (s, 3H), 4.63–4.67 (t, J = 6.86 Hz, 1H), 6.57–6.61 (d,
J = 15.76 Hz, 1H), 6.98–7.01 (d, J = 8.60 Hz, 2H), 7.38–7.42 (d,
J = 15.72 Hz, 1H), 7.52–7.54 (d, J = 8.60 Hz, 2H), 8.23–8.25 (d,
J = 8.76 Hz, 2H), 8.36–8.38 (d, J = 8.76 Hz, 2H), 8.54–8.56 (d,
J = 6.04 Hz, 1H), 13.05 (s, 1H); ESI-MS: m/z [M+H]+454.3.
5.2.30. (2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-[(p-
methoxycinnamoyl)amino]-propanamide (3f)
White solid, yield: 32.8%; mp: 188–190 °C; IR (KBr,
r
/cmꢀ1):
3420, 3270 ( NH), 1700, 1655 ( C@O), 1622 (
t
t
t
C@N); 1H NMR
(DMSO-d6): d 1.38–1.40 (d, J = 7.08 Hz, 3H), 2.37 (s, 3H), 3.79 (s,
3H), 4.63 (s, 1H), 6.57–6.61 (d, J = 15.80 Hz, 1H), 6.98–7.00 (d,
J = 8.48 Hz, 2H), 7.34–7.35 (d, J = 7.96 Hz, 2H), 7.37–7.41 (d, J =
15.84 Hz, 1H), 7.52–7.54 (d, J = 8.52 Hz, 2H), 7.82–7.84 (d,
J = 7.88 Hz, 2H), 8.53–8.55 (d, J = 6.32 Hz, 1H), 12.84 (s, 1H); ESI-
MS: m/z [M+H]+ 424.1.
5.2.31. (2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-
[(p-methoxycinnamoyl)amino]-propanamide (3g)
White solid, yield: 60.4%; mp: 144–145 °C; IR (KBr,
3407, 3280 ( NH), 1695, 1653 ( C@O), 690 (
CAS); 1H NMR (DMSO-
r
/cmꢀ1):
t
t
t
´
23. Rzeski, W.; Matysiak, J.; Kandefer-Szerszen, M. Bioorg. Med. Chem. 2007, 15,
d6): d 1.38–1.40 (d, J = 7.00 Hz, 3H), 3.79–3.83 (d, J = 14.28 Hz, 6
H), 4.61–4.65 (t, J = 6.80 Hz, 1H), 6.57–6.61 (d, J = 15.76 Hz, 1H),
6.98–7.00 (d, J = 8.52 Hz, 2H), 7.07–7.09 (d, J = 8.68 Hz, 2H), 7.37–
7.41 (d, J = 15.76 Hz, 1H), 7.52–7.54 (d, J = 8.48 Hz, 2H), 7.87–7.89
(d, J = 8.60 Hz, 2H), 8.53–8.54 (d, J = 6.24 Hz, 1H), 12.79 (s, 1H);
ESI-MS: m/z [M+H]+ 439.1.
3201.
24. Akao, Y.; Maruyama, H.; Matsumoto, K.; Ohguchi, K.; Nishizawa, K.; Sakamoto,
T.; Araki, Y.; Mishima, S.; Nozawa, Y. Biol. Pharm. Bull. 2003, 26, 1057.
25. Foroumadi, A.; Daneshtalab, M.; Shafiee, A. Arzneimittel-Forschung 1999, 49,
1035.
26. Levy, M.; Palmer, A. H. J. Biol. Chem. 1942, 146, 493.
27. Song, K. S.; Ishikawa, Y.; Kobayashi, S.; Sankawa, U.; Ebizuka, Y. Phytochemistry
1992, 31, 823.
28. Kim, H.; Burley, S. K.; Lipscomb, W. N. J. Mol. Biol. 1993, 230, 722.
29. Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Protein Eng. 1995, 8, 127.
30. Lejczak, B.; Kafarski, P.; Zygmunt, J. Biochemistry 1989, 28, 3549.
31. Baragi, V. M.; Shaw, B. J.; Renkiewicz, R. R.; Kuipers, P. J.; Welgus, H. G.;
Mathrubutham, M.; Cohen, J. R.; Rao, S. K. Matrix Biol. 2000, 19, 267.
Acknowledgements
This work was supported by National Nature Science Founda-
tion of China (Grant No. 30772654) and the Ph.D. Programs Foun-