C.-H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128233
were two series of dihydropyrazole derivatives including amide and
sulfamide. Among them, sulfamides showed better cytotoxic activity
than amides. Especially, derivative 4c exhibited the worst anticancer
activity against three cell lines at a dose of 10
μM (inhibition rate =
-
8.3%, ꢀ 0.4% and ꢀ 2.2%, respectively), derivative 4o exhibited the
best anticancer activity against three cell lines (inhibition rate = 99.8%,
9
9.8% and 100%, respectively).
To explore the structure–activity relationship (SAR) towards potent
anticancer activity of heterocyclic substituted dihydropyrazoles against
three cancer cell lines, various groups including EWG and EDG on the
phenyl ring as well as aliphatic of the derivatization functionality were
introduced. We observed that compounds containing electron-
withdrawing substituents and halogen contributed to potential anti-
tumor activity, such as CF
3
, CN, F and Br. However, EDG led to poor
Scheme 1. Design of new heterocyclic substituted dihydropyrazoles.
cytotoxic activity.
In addition, in order to contrast the anticancer effect of potent
compounds (4g, 4h, 4l, 4m and 4o) on other cancer cells, the cytotoxic
activity against human colorectal cancer cell line (HCT116), cervical
carcinoma cell line (HeLa) and lung cancer cell line (A549) were carried
out. As shown in Table 3, the result showed that five compounds dis-
influence of types of groups (electron donating and electron with-
drawing) on antitumor activity and investigate the structure–activity
relationships (SAR), various acyl and sulfonyl bearing substituents (X,
3
alkyl, alkoxy, CN and CF , etc) were introduced into the derived groups
of the title derivatives. Comparative data for new dihydropyrazole
compounds with respective to structures, formula, melting point and
yields were shown in Table 1. All of the synthesized compounds were
played selective inhibition against four cancer cell lines (IC50 < 10
Especially, compound 4o showed the best potent antitumor activity
IC50 < 3 M). We further evaluated the time-dependent efficacy of
compound 4o. Compound 4o showed max efficacy after treatment for
8 h through the evaluation of cell inhibition rate in different time
μM).
(
μ
1
13
characterized by H NMR and C NMR, and some representative com-
pounds were characterized by HRMS analysis.
4
Anticancer activity: Initially, preliminary anticancer effect of new
synthesized dihydropyrazole derivatives were evaluated on human
breast cancer cell line (HCC1806), human breast cancer cell line (MDA-
MB-231) and human lung cancer cell line (NCI-H1975) by sulforhod-
amine B (SRB). The inhibition rate of cancer cell lines under the dose of
points after treatment in HCC1806 cells (Fig. 1). Then, we carried out
the cell cycle arrest effects of compound 4o in HCC1806 cells. After
treated with compound 4o, cells of the G2/M phase were increased,
while cells of the G1 and S phase were decreased (Fig. 2).
Waf1/Cip1
Furthermore, p21
is cyclin-dependent kinase inhibitor,
1
0
μ
M were summarized in Table 2.
As shown in Table 2, some compounds showed good anticancer ac-
tivity at a dose of 10 M. In general, piperazinyl dihydropyrazole de-
which can arrest cell cycle progression in response to many stimuli,
including antitumor compounds. p21 promote cell cycle arrest in G1 and
μ
18
G2/M phase through inhibiting CDK4,6/cyclin-D and CDK2/cyclin-E,
rivatives (4) displayed the better cytotoxic activity against three cancer
cells than that of triazolyl (5) and imidazolyl products (6). Among all
derivatives, compounds 4g, 4h, 4l, 4m, 4o and 6j displayed the po-
tential cytotoxic activity against HCC1806, compounds 4g, 4o and 4j
showed good antitumor activity against MDA-MB-231, compounds 4g,
respectively. Therefore, we detected the change of p21 protein level
after treatment with indicated compound 4o for 12 h in HCC1806 cells.
Western blot showed that compound 4o induced p21 accumulation
significantly (Fig. 3).
In conclusion, a series of heterocyclic substituted dihydropyrazoles
have been designed and synthesized. We evaluated the in vitro anti-
cancer activities of derivatives against three human cancer cell lines.
The results indicated that piperazinyl dihydropyrazole compounds 4g,
4
h, 4l, 4m, 4o, 6g, 6j and 6l displayed selective inhibition activity
against NCI-H1975 (inhibition rate > 50%, respectively). Especially, the
inhibition rate of compound 4g was 93.2% against NCI-H1975, 4l was
over 90% against HCC1806 and NCI-H1975, and 4o was up to 100%
against three cancer cell lines, respectively. Besides, other compounds
displayed low cytotoxic activity against three cancer cells (inhibition
rate < 50%, respectively). The preliminary result indicated that het-
erocycle of title compounds had obvious influence on anticancer activ-
ity, and piperazine unit played a vital role in activity. Furthermore, there
4
h, 4l, 4m, 4o, 6g, 6j and 6l showed the better cytotoxic activity,
especially compound 4o displayed showed the best potent antitumor
activity. Further mechanism study demonstrated that compound 4o
could induce G2/M arrest in HCC1806 cell and p21 accumulation
significantly, which could be considered as a potent anticancer agent,
Scheme 2. Synthetic routes of dihydropyrazole derivatives. Conditions: (a) 20% KOH, EtOH, rt, 6 h; (b) K
hydrate, EtOH, reflux, 12 h; (d) RCOCl or RSO Cl, K CO
, DCM, rt, 2–5 h.
2
CO
3
, Het-NH, DMF, 110 ◦C, 12–24 h; (c) Hydrazine
2
2
3
2