Synthesis of 1,3,4-thiadiazole derivatives as aminopeptidase N inhibitors

Article abstract of DOI:10.1691/ph.2009.8630

Aminopeptidase N (APN) is a zinc-dependent ectopeptidase which plays an important role in the invasion of metastatic tumors. In this study, we report the synthesis and in vitro enzyme inhibition assay of 1,3,4-thiadiazole scaffold compounds. These new com

Full text of DOI:10.1691/ph.2009.8630

ORIGINAL ARTICLES
State Key Laboratory of Food Science and Technology1, Nanchang University; Department of Pharmacy2, NanChang Univer-
sity Medical College; Department of Cardiology3; the Second Affiliated Hospital of Nanchang University, P.R. China
Synthesis of 1,3,4-thiadiazole derivatives as aminopeptidase N inhibitors
Shao-Hua Li1,2, Gang Li2, Hui-Ming Huang2, Fang Xiong3, Xi Mai2, Bin-Hai Kuang2, Cheng-Mei Liu1, Guo-Gang Tu2
Received May 26, 2008, accepted June 30, 2008
Prof. Cheng-Mei Liu, State Key Laboratory of Food Science and Technology, Nanchang University,
Nanchang 330047, P.R. China
Dr.Guo-Gang Tu, Department of Pharmacy, NanChang University Medical College, NanChang, 330006,
P.R. China
tugg199@yahoo.com
Pharmazie 64: 67–70 (2009)
doi: 10.1691/ph.2009.8630
Aminopeptidase N (APN) is a zinc-dependent ectopeptidase which plays an important role in the inva-
sion of metastatic tumors. 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 inhibitory activities toward
APN with IC50 values in the micromolar range.
1. Introduction
(Harding et al. 1987) have been found to act as inhibitors of
APN. Synthetic APN inhibitors have been developed from
analogues of amino acids such as aminohydroxamates, ami-
noboronic acids (Shenvi et al. 1986), b-aminothiols (Four-
nie´-Zaluski et al. 1992), aminoaldehydes (Andersson et al.
1982) and aminophosphonic acids. Among them, only
b-aminothiols are both selective and relatively potent inhibi-
tors.
Inhibitors containing zinc-binding groups (ZBGs) should be
designed to inhibit the activity of APN. This class of inhibi-
tors can interact with the zinc ions of zinc-dependent metal-
loenzymes and sequentially inhibit the metastatic spread of
tumors and block the processes of tumor neovascularization
(Becker et al. 1995). There are two hydrophobic domains
beside the catalytic activity center of APN, called pockets
S1 and S01. The hydrophobic thiadiazole and aromatic ring
may interact with pockets S1 and S01. APN exhibits a broad
specificity for peptides with a N-terminal neutral or basic
amino acid such as alanine, arginine or leucine. If we link
thiadiazole with alanine and the aromatic ring, the resulting
1,3,4-thiadiazole scaffold compounds should be capable of
inhibiting the enzymatic activity of APN. So different 1,3,4-
thiadiazole derivatives were designed and synthesized
(Levy et al. 1942; Song et al. 1992).
Aminopeptidase N (APN), also known as CD13, is a wide-
spread ectopeptidase in mammalian tissues including the
central nervous system, kidney, intestine and lung which
preferentially releases neutral and basic amino acids from
the N-terminal end of peptides (Riemann et al. 1999a).
APN has been reported to play an important role in the
invasion of metastatic tumors in vitro (Saiki et al. 1993;
Menrad et al. 1993). Observations show enhanced APN le-
vels in tumor cells including melanoma (Fujii et al. 1995),
renal, pancreas (Ikeda et al. 2003), colon (Hashida et al.
2002), prostate (Ishii et al. 2001), gastric (Carl-McGrath
et al. 2004), and thyroid cancers (Kehlen et al. 2003). Tu-
mor-infiltrating T cells in lung and renal cancers are CD13-
positive (Riemann et al. 1994b). APN activity is elevated in
plasma and effusions of cancer patients (van Hensbergen
et al. 2002). APN activity on neutrophils from patients af-
fected by a rare adrenal gland tumor, adrenal pheochromo-
cytoma, is significantly increased as compared with healthy
controls (Balog et al. 2003). APN is overexpressed in acute
and chronic myeloid leukemias (Boldt et al. 1994; Tatsumi
et al. 2002) and in anaplastic large cell lymphomas (Popni-
kolov et al. 2000; Dunphy et al. 2000). Overexpression of
APN in T lymphocytes or neutrophils occurs in several in-
flammatory diseases (chronic pain, various forms of joint
effusions, rheumatoid arthritis, multiple sclerosis, systemic
sclerosis, systemic lupus erythematosus, polymyositis/der-
matomyosytis, pulmonary sarcoidosis) (Dan et al. 2003;
Abe et al. 1998). All these findings make this enzyme an
interesting target for possible therapeutic applications,
which require the development of potent and selective inhi-
bitors. APN inhibition by bestatin leads to a loss of moti-
lity. Likewise, CD13 antibodies inhibit cell growth and cell
motility (Antczak et al. 2001; Lohn et al. 2002).
2. Investigations, results and discussion
We prepared analogues substituting the phenyl ring in po-
sition 5 of the 1,3,4-thiadiazole moiety with various halo-
gens (1b–d, 2b–d, 3b–d), electron-withdrawing groups
(1e, 2e, 3e), and moderately electron-donating groups (1f–g,
2f–g, 3f–g) and assessed the effects that these changes had
on APN and MMP-2 inhibitory activity. All the inhibition
results are summarized in the Table. The inhibition results
showed that most of the target compounds display excellent
potency toward APN with IC50 values lying in a micromolar
´
Natural products including bestatin, actinonin (Grujic et al.
2002), MA-387A and B (Bauvois et al. 2006) and amastatin
Pharmazie 64 (2009) 2
67

ORIGINAL ARTICLES
Table: In vitro enzyme assay results for compounds 1–3 and bestatin
Structure
Compounds
R
IC50a/mM
1a
1b
1c
1d
1e
1f
––H
––F
––Cl
––Br
––NO2
––CH3
––OCH3
170.1 ꢃ 12.8
190.5 ꢃ 16.3
287.5 ꢃ 12.8
308.9 ꢃ 17.6
317.8 ꢃ 118.3
117.8 ꢃ 17.5
69.8 ꢃ 7.1
141.6 ꢃ 12.9
169.0 ꢃ 14.6
205.8 ꢃ 17.9
296.6 ꢃ 18.6
309.1 ꢃ 10.8
107.0 ꢃ 17.5
39.9 ꢃ 4.5
1a–g
1g
2a
2b
2c
2d
2e
2f
––H
––F
––Cl
––Br
––NO2
––CH3
––OCH3
2a–g
3a–g
2g
3a
3b
3c
3d
3e
3f
––H
––F
––Cl
––Br
––NO2
––CH3
––OCH3
373.2 ꢃ 14.3
115.3 ꢃ 12.4
96.4 ꢃ 3.2
107.3 ꢃ 18.1
167.8 ꢃ 12.6
337.8 ꢃ 15.9
48.8 ꢃ 8.0
3g
Bestatin
8.5 ꢃ 0.6
a
Values are means of three experiments, standard deviation is given
MS were determined on an Aglient-1100 series LC/MSD trap spectro-
meter. IR were recorded on a FTIR-8400 spectrometer. Melting points
were determined on a electrothermal melting point apparatus and are uncor-
rected. 1H NMR spectra were obtained on a Bruker-400. The chemical
shifts are expressed in d values (parts per million) relative to tetramethylsi-
lane (TMS) as internal standard. Significant 1H NMR data are reported in
the following order: multiplicity (s, singlet; d, doublet; t, triplet; m, multi-
plet) number of protons.
level. Comparing phenylamide and benzylamide analogues
(1a–g, 2a–g), halogens and electron-withdrawing groups
attached to the 4-position of the phenyl ring decreased the
potency against APN. However, The electron-donating
group (1f–g, 2f–g) improved the inhibitory activity against
APN. Substitution by electron-donating substituents pro-
duces an electron-sufficient phenyl ring, which may improve
aryl-aryl stacking interactions with aromatic side chains in
the active site of APN. The more active compound 2g was
introduced the flexible benzylamide which may improve the
interaction with the active site of APN. Halogens, electron-
withdrawing groups and electron-donating groups of benzyl-
sulfonamide analogues all increased the potency against
APN. In addition, all the target compounds show inactivity
toward MMP-2 which is a zinc-dependent endopeptidase as-
sociated with the tumorigenic process. MMP-2 is a zinc-
dependent endopeptidase that can cut the peptide to parts
from the specific amino acid residue of the peptide. How-
ever, APN is a membrane-bound zinc exopeptidase that
catalyzes the removal of the NH-terminal amino acid from
the peptide. Due to the structural differences between
MMP-2 and APN, there were different structural require-
ments for their respective inhibitors. So, most of com-
pounds show more inhibitory activities on APN than on
MMPs due to the recognition of APN.
3.1.1. General procedure for the preparation of 1,3,4-thiadiazole derivatives.
Synthesis of (2R)-N-[5-phenyl-1,3,4-thiadiazol-2-yl]-2-[(benzoyl)-amino]-
propanamide ð1aÞ
N,N-Dicyclohexylcarbodiimide (6.2 mmol) was added to a cooled solution
of N-benzoyl- D-alanine (6.0 mmol) and N-hydroxystinimide (6.0 mmol) in
freshly distillation dioxane (30 ml). The reaction mixture was stirred over-
night at room temperature. The insoluble material was filtered off and
washed with cold dioxane. 5-Phenyl-1,3,4-thiadiazol-2-Amino (5.6 mmol)
was added to the filtrate and the reaction mixture was stirred for 48 h at
room temperature. The solvent was removed under reduced pressure. The
residual was dissolved in EtOAc and the insoluble material was filtered
off. The filtrate was washed successively with saturated Na2CO3 solution
(20 ml, ꢀ 3), water (20 ml, ꢀ 1), 0.1 M HCl (20 ml, ꢀ 3) and water
(20 ml, ꢀ 1). The organic layer was evaporated in vacuo, the residual was
recrystallized from methanol/acetonitrile (5 : 1) and dried to afford the tar-
get compound as white solid 1.27 g, yield: 64.5%; m.p.: 220–222 ꢁC; IR
(KBr, s/cmꢂ1): 3436, 3243 (uNH), 1700, 1654 (uC¼O), 689 (uCꢂS), 1638
(uC¼N); 1H NMR (DMSO-d6): d 1.49–1.51 (d, J ¼ 7.08 Hz, 3H), 4.69–
4.76 (m, 1H), 7.48–7.59 (m, 6H), 7.88–7.95 (t, J ¼ 12.8Hz, 4H), 8.82–
8.84 (d, J ¼ 6.04 Hz, 1H), 12.86 (s, 1H); ESI-MS: m/z [M þ H]þ 353.3.
3.1.2. (2R)-N-[5-(4-Fluorophenyl)-1,3,4- thiadiazol-2-yl]-2-
[(benzoyl)amino]- propanamide ð1bÞ
In conclusion, we have developed a new series of potent
APN inhibitors. Most of the compounds have potent inhibi-
tory activities toward APN and could be used as lead com-
pounds for the development of low molecular-weight pepti-
domimetic APN inhibitors. The critical point in designing
selective inhibitors is to optimize the recognition of APN.
Yield: 60.2%; m.p.: 170–171 ꢁC; IR (KBr, s/cmꢂ1): 3275, 3166 (uNH),
1701, 1655 (uC¼O), 694 (uCꢂS), 1635 (uC¼N); 1H NMR (DMSO-d6): d
1.47–1.49 (d, J ¼ 7.12 Hz, 3H), 4.68–4.72 (t, J ¼ 6.76 Hz, 1H), 7.36–
7.40 (t, J ¼ 8.74 Hz, 2H), 7.48–7.51 (t, J ¼ 7.36 Hz, 2H), 7.55–7.58 (t,
J ¼ 6.94 Hz, 1H), 7.91–7.93 (d, J ¼ 8.04 Hz, 2H), 7.98–8.02 (t, J ¼ 6.90
Hz, 2H), 8.84–8.85 (d, J ¼ 5.84 Hz, 1H), 12.89 (s, 1H); ESI-MS: m/z
[M þ H]þ 371.1.
3. Experimental
3.1.3. (2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(benzoyl)amino]-
3.1. Chemistry
propanamide ð1cÞ
Unless otherwise noted, materials were obtained from commercial suppli-
ers and used without further purification. All the solvents except DMF
have been distilled before use. All reactions were monitored by TLC on
0.25 mm silica gel plates (60GF-254) and visualized with UV light. Col-
umn chromatography was performed on silica gel (200–300 mesh). ESI-
Yield: 69.8%; m.p.: 216–217 ꢁC; IR (KBr, s/cmꢂ1): 3333, 3159 (uNH),
1698,1643 (uC¼O), 691 (uCꢂS), 1630 (uC¼N); 1H NMR (DMSO-d6): d
1.48–1.50 (d, J ¼ 7.12 Hz, 3H), 4.69–4.73 (t, J ¼ 6.72 Hz, 1H), 7.47–
7.60 (m, 5H), 7.92–7.97 (t, J ¼ 9.28 Hz, 4H), 8.87–8.88 (d, J ¼ 5.96 Hz,
1H), 12.95 (s, 1H); ESI-MS: m/z [M þ H]þ 387.1.
68
Pharmazie 64 (2009) 2

ORIGINAL ARTICLES
3.1.4. (2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-[(benzoyl)amino]-
(t, J ¼ 6.84 Hz, 1H), 7.22–7.32 (m, 5H), 7.33–7.35 (d, J ¼ 7.96 Hz,
2H), 7.82–7.84 (d, J ¼ 7.96 Hz, 2H), 8.62–8.64 (d, J ¼ 6.28 Hz, 1H),
12.79 (s, 1H); ESI-MS: m/z [M þ H]þ 412.3.
propanamide ð1dÞ
Yield: 42.5%; m.p.: 191–193 ꢁC; IR (KBr, s/cmꢂ1): 3328, 3166 (uNH),
1702, 1648 (uC¼O), 692 (uCꢂS); 1H NMR (DMSO-d6): d 1.47–1.49 (d,
J ¼ 7.12 Hz, 3H), 4.69–4.72 (t, J ¼ 6.78 Hz, 1H), 7.47–7.74 (m, 5H),
7.88–7.90 (d, J ¼ 8.44 Hz, 2H), 7.92–7.93 (d, J ¼ 7.44 Hz, 2H), 8.86–
8.88 (d, J ¼ 6.04 Hz, 1H), 12.95 (s, 1H); ESI-MS: m/z [M þ H]þ 432.9.
3.1.13. (2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)-
amino]-propanamide ð2fÞ
Yield: 32.1%; m.p.: 185–186 ꢁC; IR (KBr, s/cmꢂ1): 3421, 3301 (uNH),
1699, 1647 (uC¼O), 695 (uCꢂS), 1624 (uC¼N); 1H NMR (DMSO-d6): d
1.34–1.36 (d, J ¼ 7.12 Hz, 3H), 2.37 (s, 3H), 3.54 (s, 2H), 4.48–4.51 (t,
J ¼ 6.84 Hz, 1H), 7.22–7.32 (m, 5H), 7.33–7.35 (d, J ¼ 7.96 Hz, 2H),
7.82–7.84 (d, J ¼ 7.96 Hz, 2H), 8.62–8.64 (d, J ¼ 6.28 Hz, 1H), 12.79
(s, 1H); ESI-MS: m/z [M þ H]þ 381.1.
3.1.5. (2R)-N-[5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(benzoyl)amino]-
propanamide ð1eÞ
Yield: 20.7%; m.p.: 225–226 ꢁC; IR (KBr, s/cmꢂ1): 3415, 3333 (uNH),
1695, 1650 (uC¼O), 693 (uCꢂS), 1628 (uC¼N), 1517 (uasNO2), 1347 (usNO2);
1H NMR (DMSO-d6): d 1.49–1.50 (d, J ¼ 7.20 Hz, 3H), 4.70–4.73 (t,
J ¼ 6.74 Hz, 1H), 7.48–7.52 (t, J ¼ 7.44 Hz, 2H), 7.56–7.57 (d, J ¼ 7.24
Hz, 1H), 7.92–7.95 (t, J ¼ 6.46 Hz, 2H), 8.23–8.25 (d, J ¼ 8.76 Hz,
2H), 8.35–8.38 (d, J ¼ 8.80 Hz, 2H), 8.89–8.90 (d, J ¼ 6.04 Hz, 1H),
13.10 (s, 1H); ESI-MS: m/z [M þ H]þ 398.1.
3.1.14. (2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)-
amino]-propanamide ð2gÞ
Yield: 38.8%; m.p.: 142–143 ꢁC; IR (KBr, s/cmꢂ1): 3329, 3165 (uNH),
1690, 1670 (uC¼O), 695 (uCꢂS); 1H NMR (DMSO-d6): d 1.34–1.36 (d,
J ¼ 7.08 Hz, 3H), 3.46–3.53 (t, J ¼ 15.36 Hz, 2H), 3.83 (s, 3H), 4.48–
4.51 (t, J ¼ 6.80 Hz, 1H), 7.07–7.09 (d, J ¼ 8.68 Hz, 2H), 7.22–7.32 (m,
5H), 7.87–7.89 (d, J ¼ 8.60 Hz, 2H), 8.61–8.63 (d, J ¼ 6.24 Hz, 1H),
12.75 (s, 1H); ESI-MS: m/z [M þ H]þ 397.1.
3.1.6. (2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-[(benzoyl)amino]-
propanamide ð1fÞ
Yield: 29.2%; m.p.: 189–190 ꢁC; IR (KBr, s/cmꢂ1): 3301, 3165 (uNH),
1703, 1655 (uC¼O), 691 (uCꢂS); 1H NMR (DMSO-d6): d 1.49–1.51 (d,
J ¼ 7.20 Hz, 3H), 2.41 (s, 3H), 4.71–4.74 (t, J ¼ 6.84 Hz, 1H), 7.33–
7.35 (d, J ¼ 8.04 Hz, 2H), 7.48–7.59 (m, 3H), 7.82–7.84 (d, J ¼ 8.12
Hz, 2H), 7.93–7.95 (t, J ¼ 4.30 Hz, 2H), 8.82–8.84 (d, J ¼ 6.32 Hz,
1H), 12.83 (s, 1H); ESI-MS: m/z [M þ H]þ 365.2.
3.1.15. (2R)-N-[5-phenyl-1,3,4- thiadiazol-2-yl]-2-[(phenylsulfonyl)amino]-
propanamide ð3aÞ
Yield: 25.8%; m.p.: 258–260 ꢁC; IR (KBr, s/cmꢂ1): 3447, 3175 (uNH),
1700 (uC¼O), 1321 (uasO¼S¼O), 1147 (usO¼S¼O), 689 (uCꢂS); 1H NMR
(DMSO-d6): d 1.24–1.25 (d, J ¼ 7.04 Hz, 3H), 4.16–4.23 (m, 1H),
7.51–7.56 (m, 6H), 7.79–7.82 (dd, J1 ¼ 1.36 Hz, J2 ¼ 7.72 Hz, 2H),
7.94–7.92 (m, 2H), 8.42–8.40 (d, J ¼ 7.96Hz, 1H), 12.72 (s, 1H); ESI-
MS: m/z [M þ H]þ 389.2.
3.1.7. (2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-[(benzoyl)amino]-
propanamide ð1gÞ
Yield: 40.3%; m.p.: 155–156 ꢁC; IR (KBr, s/cmꢂ1): 3407, 3298 (uNH),
1699, 1660 (uC¼O), 692 (uCꢂS); 1H NMR (DMSO-d6): d 1.47–1.49 (d,
J ¼ 7.12 Hz, 3H), 3.83 (s, 3H), 4.69 (s, 1H), 7.07–7.09 (d, J ¼ 8.68 Hz,
2H), 7.48–7.51 (t, J ¼ 7.36 Hz, 2H), 7.55–7.57 (d, J ¼ 7.08 Hz, 1H),
7.86–7.89 (d, J ¼ 8.68 Hz, 2H), 7.92–7.93 (d, J ¼ 7.36 Hz, 2H), 8.84–
8.86 (d, J ¼ 6.08 Hz, 1H), 12.82 (s, 1H); ESI-MS: m/z [M þ H]þ 383.1.
3.1.16. (2R)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylsulfo-
nyl)amino]-propanamide ð3bÞ
Yield: 38.6%; m.p.: 199–200 ꢁC; IR (KBr, s/cmꢂ1): 3423, 3182 (uNH),
1696 (uC¼O), 1323 (uasO¼S¼O), 1149 (usO¼S¼O), 690 (uCꢂS); 1H NMR
(DMSO-d6): d 1.21–1.23 (d, J ¼ 7.04 Hz, 3H), 4.15–4.18 (t, J ¼ 7.32
Hz, 1H), 7.36–7.41 (t, J ¼ 8.76 Hz, 2H), 7.50–7.56 (dd, J1 ¼ 7.92 Hz,
J2 ¼ 15.36 Hz, 3H), 7.77–7.79 (t, J ¼ 3.94 Hz, 2H), 7.97–8.01 (dd,
J1 ¼ 5.44 Hz, J2 ¼ 8.56 Hz, 2H), 8.43–8.45 (d, J ¼ 7.96 Hz, 1H), 12.76
(s, 1H); ESI-MS: m/z [M þ H]þ 407.0.
3.1.8. (2R)-N-[5-phenyl-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)amino]-pro-
panamide ð2aÞ
Yield: 14.0%; m.p.: 245–247 ꢁC; IR (KBr, s/cmꢂ1): 3242, 3184 (uNH),
1712, 1646 (uC¼O), 688 (uCꢂS); 1H NMR (DMSO-d6): d 1.36–1.39 (d,
J ¼ 7.12 Hz, 3H), 3.48–3.55 (t, J ¼ 15.08 Hz, 2H), 4.49–4.56 (m, 1H),
7.20–7.24 (m, 1H), 7.27–7.32 (m, 4H), 7.53–7.54 (t, J ¼ 2.78 Hz, 3H),
7.93–7.96 (m, 2H), 8.59–8.61 (d, J ¼ 6.32 Hz, 1H), 12.80 (s, 1H); ESI-
MS: m/z [M þ H]þ 367.3.
3.1.17. (2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylsulfonyl)-
amino]-propanamide ð3cÞ
Yield: 80.1%; m.p.: 226–228 ꢁC; IR (KBr, s/cmꢂ1): 3446, 3196 (uNH),
1703 (uC¼O), 1315 (uasO¼S¼O), 1151 (usO¼S¼O), 691 (uCꢂS); 1H NMR
(DMSO-d6): d 1.21–1.23 (d, J ¼ 7.08 Hz, 3H), 4.14–4.21 (m, 1H),
7.50–7.57 (m, 5H), 7.77–7.80 (dd, J1 ¼ 1.40 Hz, J2 ¼ 7.56 Hz, 2H),
7.91–7.93 (dd, J1 ¼ 1.84 Hz, J2 ¼ 6.80 Hz, 2H), 8.38–8.40 (d, J ¼ 8.00
Hz, 1H), 12.74 (s, 1H); ESI-MS: m/z [M þ H]þ 423.1.
3.1.9. (2R)-N-[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)-
amino]-propanamide ð2bÞ
Yield: 41.8%; m.p.: 140–141 ꢁC; IR (KBr, s/cmꢂ1): 3397, 3309 (uNH),
1700, 1650 (uC¼O), 696 (uCꢂS); 1H NMR (DMSO-d6): d 1.34–1.36 (d,
J ¼ 7.12 Hz, 3H), 3.50 (s, 2H), 4.48–4.51 (t, J ¼ 6.94 Hz, 1H), 7.22–
7.32 (m, 5H), 7.36–7.41 (t, J ¼ 8.74 Hz, 2H), 7.99–8.02 (t, J ¼ 6.95 Hz,
2H), 8.63–8.64 (d, J ¼ 6.20 Hz, 1H), 12.84 (s, 1H); ESI-MS: m/z
[M þ Na]þ 407.1.
3.1.18. (2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylsulfonyl)-
amino]-propanamide ð3dÞ
Yield: 22.8%; m.p.: 240–241 ꢁC; IR (KBr, s/cmꢂ1): 3430, 3185 (uNH),
1708 (uC¼O), 1315 (uasO¼S¼O), 1151 (usO¼S¼O), 691 (uCꢂS); 1H NMR
(DMSO-d6): d 1.22–1.23 (d, J ¼ 6.96 Hz, 3H), 4.16–4.19 (t, J ¼ 7.30
Hz, 1H), 7.50–7.55 (dd, J1 ¼ 7.04 Hz, J2 ¼ 14.44 Hz, 3H), 7.72–7.74 (d,
J ¼ 8.36 Hz, 2H), 7.78–7.80 (d, J ¼ 6.64 Hz, 2H), 7.86–7.89 (d,
J ¼ 8.36 Hz, 2H), 8.44–8.46 (d, J ¼ 7.96 Hz, 1H), 12.80 (s, 1H); ESI-
MS: m/z [M þ H]þ 468.9.
3.1.10. (2R)-N-[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)-
amino]-propanamide ð2cÞ
Yield: 35.9%; m.p.: 120–121 ꢁC; IR (KBr, s/cmꢂ1): 3418, 3175 (uNH),
1703, 1650 (uC¼O), 695 (uCꢂS); 1H NMR (CDCl3): d 1.55–1.57 (d,
J ¼ 7.40 Hz, 3H), 3.53–3.61 (dd, J1 ¼ 15.08 Hz, J2 ¼ 17.40 Hz, 2H),
4.80–4.87 (m, 1H), 7.09–7.15 (m, 5H), 7.48–7.50 (d, J ¼ 8.48 Hz, 2H),
7.77–7.80 (dd, J1 ¼ 1.72 Hz, J2 ¼ 6.96 Hz, 2H), 7.89–7.91 (d, J ¼ 6.88
Hz, 1H), 13.05 (s, 1H); ESI-MS: m/z [M þ H]þ 401.2.
3.1.19. (2R)-N-[5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylsulfonyl)-
amino]-propanamide ð3eÞ
Yield: 46.8%; m.p.: 206–208 ꢁC; IR (KBr, s/cmꢂ1): 3428, 3198 (uNH),
1695 (uC¼O),1320 (uasO¼S¼O), 1148 (usO¼S¼O), 691 (uCꢂS),1523 (uasNO2),
1345 (usNO2); 1H NMR (DMSO-d6): d 1.23–1.25 (d, J ¼ 6.96 Hz, 3H),
4.17–4.20 (t, J ¼ 7.32 Hz, 1H), 7.51–7.56 (dd, J1 ¼ 7.16 Hz, J2 ¼ 14.64
Hz, 3H), 7.78–7.80 (d, J ¼ 6.68 Hz, 2H), 8.21–8.23 (d, J ¼ 8.68 Hz,
2H), 8.36–8.38 (d, J ¼ 8.68 Hz, 2H), 8.46–8.48 (d, J ¼ 8.08 Hz, 1H),
12.95 (s, 1H); ESI-MS: m/z [M þ H]þ 434.3.
3.1.11. (2R)-N-[5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)-
amino]-propanamide ð2dÞ
Yield: 26.6%; m.p.: 161–162 ꢁC; IR (KBr, s/cmꢂ1): 3366, 3262 (uNH),
1695, 1648 (uC¼O), 695 (uCꢂS); 1H NMR (DMSO-d6): d 1.35–1.36 (d,
J ¼ 7.12 Hz, 3H), 3.50 (s, 2H), 4.48–4.52 (t, J ¼ 6.84 Hz, 1H), 7.22–
7.32 (m, 5H), 7.72–7.75 (d, J ¼ 8.40 Hz, 2H), 7.88–7.91 (d, J ¼ 8.40
Hz, 2H), 8.64–8.65 (d, J ¼ 6.24 Hz, 1H), 12.88 (s, 1H); ESI-MS: m/z
[M þ H]þ446.9.
3.1.20. (2R)-N-[5-(4-Methylphenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylsulfonyl)-
amino]-propanamide ð3fÞ
Yield: 31.5%; m.p.: 215–216 ꢁC; IR (KBr, s/cmꢂ1): 3436, 3164 (uNH),
1699 (uC¼O), 1315 (uasO¼S¼O), 1151 (usO¼S¼O), 691 (uCꢂS); 1H NMR
(DMSO-d6): d 1.21–1.23 (d, J ¼ 6.80 Hz, 3H), 2.37 (s, 3H), 4.15–4.19
(t, J ¼ 7.12 Hz, 1H), 7.34–7.36 (d, J ¼ 7.80 Hz, 2H), 7.52–7.56 (t,
J ¼ 6.98 Hz, 3H), 7.78–7.82 (t, J ¼ 7.66 Hz, 4H), 8.41–8.43 (d,
J ¼ 7.72Hz, 1H), 12.70 (s, 1H); ESI-MS: m/z [M þ H]þ 403.0.
3.1.12. (2R)-N-[5-(4-Nitrorophenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylacetyl)-
amino]-propanamide ð2eÞ
Yield: 44.3%; m.p.: 218–220 ꢁC; IR (KBr, s/cmꢂ1): 3405, 3277 (uNH),
1699, 1651 (uC¼O), 690 (uCꢂS), 1521 (uasNO2), 1344 (usNO2); 1H NMR
(DMSO-d6): d 1.34–1.36 (d, J ¼ 7.12 Hz, 3H), 3.54 (s, 2H), 4.48–4.51
Pharmazie 64 (2009) 2
69

ORIGINAL ARTICLES
3.1.21. (2R)-N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-[(phenylsul-
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fonyl)amino]-propanamide ð3gÞ
Yield: 10.9%; m.p.: 237–238 ꢁC; IR (KBr, s/cmꢂ1): 3445, 3205 (uNH),
1700 (uC¼O), 1304 (uasO¼S¼O), 1151 (usO¼S¼O), 691 (uCꢂS); 1H NMR
(DMSO-d6): d 1.21–1.23 (d, J ¼ 6.96 Hz, 3H), 3.83 (s, 3H), 4.16 (s,
1H), 7.08–7.10 (d, J ¼ 8.64 Hz, 2H), 7.51–7.5 (dd, J1 ¼ 7.72 Hz,
J2 ¼ 15.20 Hz, 3H), 7.78–7.79 (d, J ¼ 6.76 Hz, 2H), 7.85–7.87 (d,
J ¼ 8.60 Hz, 2H), 8.41–8.43 (d, J ¼ 7.80 Hz, 1H), 12.67 (s, 1H); ESI-
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another 60 min. The resulting solutions were measured under 405 nm to gain
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[OD405(100%) – OD405(compound)]/[OD405(100%) – OD405(blank)] ꢄ 100%.
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compound and the enzyme were dissolved in sodium borate buffer (pH 8.5,
50 mM), and incubated at 37 ꢁC for 30 min. The substrate was added and
incubated at 37 ꢁC for another 60 min. Then 0.03% picrylsulfonic acid so-
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resulting solutions were measured under 450 nm to gain OD450 values,
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