Y. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4125–4128
4127
Figure 2. Characterization of 5k to PTP1B. (A) Time-independent inhibition of PTP1B by 5k. (B) Mixed inhibition of 5k shown by Lineweaver–Burk plot. (C) At various fixed
concentrations of 5k the initial velocity was determined with various concentrations of pNPP.
catalysis to give the desired compounds 4a–4q and 5a–5n
Table 1). All these compounds, 4a–4q and 5a–5n are reported
(4l and 5j) showed more potent inhibitory activities against PTP1B
than those with electron-donating substituents in the benzamide
(
for the first time. The synthetic procedures for the preparation of
intermediate compounds 1–3 have been published previously.
portion, and one electron-withdrawing NO
on the A-ring have more potent inhibitory activities than those
with two electron-withdrawing NO group (5a, 5d and 5k).
2
group (4b, 4e and 4l)
9
The target compounds 4–5 were obtained in 35–72% yields. All of
synthetic target compounds gave satisfactory elementary analyti-
cal and spectroscopic data, and they were consistent with the
assigned structures.
We evaluated the inhibitory activity of the synthesized target
compounds 4a–q and 5a–n against Cdc25B and PTP1B. The results
was summarized in Table 1. As illustrated in Table 1, most com-
pounds showed potential inhibitory activities for Cdc25B and
PTP1B. Among them, compound 4f and 5b displayed the most
2
We investigated the selectivity of 31 synthesized target com-
pounds against other PTPs (TCPTP, SHP-1, SHP-2, LAR and Cdc25B).
The assays were performed according to procedures described pre-
1
9,20
viously.
The results was summarized in Table 1. As shown in
Table 1, the target compounds were discovered to be selective
inhibitors against PTP1B and Cdc25B.
A kinetic study of compound 5k was performed in order to
identify the inhibitory mechanism of target compounds (Fig. 2).
The assays were performed according to procedures described pre-
potent inhibitory activity for Cdc25B (IC50 = 1.24
.18 g/mL), and the inhibitory activity is close to the positive
control sodium orthovanadate (IC50 = 0.93 g/mL). Compound 4l
showed the most potent inhibitory activity for PTP1B (IC50 = 0.85
g/mL), and the inhibitory activity is equal to the positive control
oleanolic acid (IC50 = 0.85 g/mL).
lg/mL and
1
9
1
l
viously. As shown in Figure 2A, 5k demonstrated a time-inde-
pendent inhibition of PTP1B, and the result indicated 5k was a
fast-binding inhibitor of PTP1B. The time-independent inhibition
of 5k toward PTP1B may also exclude that 5k is a nonspecific
inhibitor, because nonspecific inhibitors always show time-
dependent behavior and steep inhibition curve. We further deter-
mined the inhibition modality of 5k which inhibited PTP1B with
the typical characteristics of a mixed inhibitor, as indicated by
l
l
l
To explore the structure–activity relationship and optimize the
structures of 2,5-disubstituited-1,3,4-thiadiazole amide deriva-
tives, we varied the substituent on phenyl ring at 5-position and
the substituent on 2-position of 1,3,4-thiadiazole ring to synthe-
size a series of target compounds 4a–q and 5a–n (Scheme 1) and
evaluate their inhibitory activity for Cdc25B and PTP1B. As shown
in Table 1, the activity of the tested compounds could be correlated
to structure variation and modifications. By investigating, it was
revealed that different acids chloride substitutes led to different
inhibitory activity, and the potency order of inhibition Cdc25B
was benzoic acid > naphthylacetic acid > furoic acid > nicotinic
acid, while the potency order of inhibition PTP1B was benzoic
acid > naphthylacetic acid > nicotinic acid > furoic acid.
Regarding these compounds (4a–m and 5a–l) with benzoic acid
substituents, structure–activity relationships in these compounds
demonstrated that compounds with para electron-donating sub-
stituents (4f and 5b) showed more potent inhibitory activities
against Cdc25B than those with electron-withdrawing substituents
in the benzamide portion. A comparison of the substituents on the
m
increased k values and reduced Vmax values when the inhibitor
concentration was increased (Fig. 2C). Meanwhile, the result of
the Lineweaver–Burk plot confirmed 5k as a mixed inhibition of
PTP1B for intersecting at second quadrant of a nest of lines with
increased inhibitor concentration (Fig. 2B).
In conclusion, we synthesized a series of 2,5-disubstituted-
1,3,4-thiadiazole amide derivatives and evaluated for their inhibi-
tory activity against Cdc25B and PTP1B. Varying the substituents
(X) at the A-ring showed that the presence of electron-withdrawing
group at the 5-position is important for inhibitory activities against
Cdc25B and PTP1B, with 4-methoxy/4-methyl substituted
compound 4f and 5b being the most potent with IC50 values of
1.24 and 1.18
compound 4l and 5k being the most potent with IC50 values of
0.85 and 1.36 g/mL against PTP1B. The results of selectivity
lg/mL against Cdc25B, with 3-nitro substituted
l
experiments showed that the target compounds were selective
inhibitors against PTP1B and Cdc25B. Enzyme kinetic experiments
demonstrated that compound 5k was a specific inhibitor with the
typical characteristics of a mixed inhibitor.
A-ring demonstrated that two electron-withdrawing NO
4b and 5a) have slightly improved inhibitory activity. On the
contrary, the compounds with electron-withdrawing substituents
2
group
(