G Model
CCLET-6133; No. of Pages 5
Q. Sun, Q. Dai, C. Zhang et al.
Chinese Chemical Letters xxx (xxxx) xxx–xxx
targeting DNMT and HDAC simultaneously could be an effective
strategy to develop antitumor agents. However, the DNMT
inhibitory potency of 15a (70% inhibition rate against DNMT1 at
group and a linker. Additionally, uracil and adenine hydroxamic
acid derivatives were also constructed as compared compounds.
We initially constructed a triazole cycle to connect nucleoside
bases and hydroxamic acid group for the sake of convenient
synthesis (Fig. 1C). Unfortunately the obtained compounds (e.g.,
Cpd-101) showed too strong water solubility with unfavorable
cLogP values to be used in medical or biological applications. To
circumvent this issue, we then optimized the linker by inserting a
benzene group, which could also be a part of the cap to compose
HDACi. The resulted compounds 201–208 showed more favorable
cLogP values than compounds 101–105 (Fig. 1C). We then
synthesized the designed compounds following procedures
showed in Scheme 1. The detailed synthesis and characterization
of final products 101–105 and 201–208 were documented in
Supporting information.
To get potential antitumor agents, we firstly evaluated the
inhibitory activities of our compounds against tumor cells
proliferation, using HDAC inhibitor SAHA and DNMT inhibitor
SGI1027 as positive compounds. The inhibition rates of tumor cells
treating with our compounds were primarily tested. The results
indicated that most of our compounds (especially 101–105, 201
and 202) showed pretty weak proliferation inhibitory activity
(inhibition rates less than 50%) in solid tumor cells, such as A549,
HCT116, HeLa and MDA-MB-231, but showed better inhibitory
activity in leukemia cells, such as K562 and U937 (Fig. S1 in
Supporting information). The weak activity of our compounds
101–105, 201 and 202 might be caused by the too strong
hydrophilic property to enter tumor cells. We further evaluated
the IC50 values of compounds 203–208, which showed > 50%
inhibition rate in tumor cells K562, U937 and HCT116. As shown in
Table 1, most compounds showed more potent inhibitory activity
against U937 than K562 and HCT116. Compounds 204–208 could
significantly inhibited U937 cells proliferation with IC50 values less
100 mmol/L) and the HDAC inhibitory activity of C02S [HDAC1 half
maximal inhibitory concentration (IC50) = 4.16
further optimized.
mmol/L] need to be
To advance more potent DNMTand HDAC dual inhibitors, in this
report we designed and synthesized a novel series of hydroxamic
acid derivatives of nucleoside bases (e.g., cytosine, uracil and
adenine) utilizing fragment-based rational drug design strategy.
The unique pharmacologic properties of representative compound
204 and its potential anticancer potency through epigenetic
reprogramming were documented.
Generally, HDACi could be divided into different classes (e.g.,
hydroxamates, cyclic peptides, benzamides and fatty acids)
according to their chemical structures. HDACi possessing hydroxa-
mic acid moiety have in common a well-admitted pharmacophore
model andsharesimilarcharacteristicsconsistingof threegroups:a
cap group, which occludes the entrance of the active site pocket; a
zinc-binding group (ZBG), which chelates the zinc ion in the active
site and is required for catalytic function; and a linker, which
connects the cap group and ZBG [26–28]. Now three hadroxamates
(i.e., vorinostat, belinostat and panobinostat) have been approved
by the US FDA (Fig. 1A). On the other side, DNMTi usually divided
intonucleoside analogues (e.g., azacytidineand decitabine) (Fig.1B)
and non-nucleoside analogues (e.g., RG108, SGI1027 and DC-517)
according to their chemical structures and functional mechanisms.
Nucleoside analogues exhibited potent DNMT inhibitory potency
and significant antitumor activity, while they could also cause
advance side effects by incorporating into DNA or RNA.
Given that cytosine is a component part of the substrate of
DNMT, and that the approved DNMTi azacytidine and decitabine
are derivatives of cytosine, herein we intended to develop dual
DNMT and HDAC inhibitors by incorporating the hydroxamic acid
group to cytosine through a proper linker unit (Fig. 1C). We
proposed that the cytosine groups of our designed compounds
could occupy the substrate binding domain of DNMT, therefore the
compounds might possess DNMT inhibitory activity and cause less
side effects than nucleoside analogues for they would not
incorporate into DNA/RNA. Besides, the compounds might display
HDAC inhibitory activity as they contain the three pharmacophore
characteristics of HDAC inhibitors: a cap group, a zinc-binding
than 10 mmol/L.
As compounds 203–208 showed good antitumor proliferation
activity, we then evaluated the DNMTand HDAC inhibitory potency
of compounds 201–208. As shown in Table 2, compounds 201–204
exhibited potent inhibitory activity against DNMT1 (inhibition
rates approximate to 90%) and barely inhibited DNMT3A/3B
(inhibition rates less than 20%) at 50 mmol/L. These results
indicated the practicability of our strategy to develop cytosine
derivatives as DNMT1 inhibitors, and that the length of the linker
has a relatively small impact for DNMT1 inhibitory potency.
Besides, 5-flurine-substituted cytosine could be acceptable as 206
exhibited comparable DNMT1 inhibition activity. However, ana-
logues of 5-methyl-substituted cytosine, uracil and adenine (i.e.,
205, 207 and 208, respectively) caused remarkable decrease of
inhibitory activity against DNMT1.
Moreover, our compounds significantly inhibited HDAC1/6 in
nanomolar concentrations. For compounds 201–204, the HDAC
inhibitory activities improved successively with the increase of
linker length of compounds, indicating that the length of linker is
critical for HDAC inhibitory potency. Compounds 204–208
presented similar HDAC1/6 inhibition activities, suggesting that
different nucleoside bases are tolerable as the cap group for the
design of HDAC inhibitors.
Given that compound 204 displayed good DNMT1 and HDAC1/6
inhibitory potency simultaneously, we chose it as a representative
compound for further biological evaluations and subsequently
tested its DNMT1 inhibitory activity at different concentrations.
The results indicated that 204 inhibited DNMT1 with IC50 value of
6.39
potent than the reported DNMT inhibitor RG-108 (IC50 = 390
L) [29] and SGI-1027 (IC50 = 35 mol/L) [30].
Since 204 exhibited potent DNMT1 and HDAC inhibitory
potency in vitro enzymatic inhibition evaluation, we further
m
mol/L (Fig. S2 in Supporting information), which is more
m
mol/
m
Fig. 1. (A) Approved hydroxamates as HDAC inhibitors; (B) Reported nucleoside
analogues as DNMT inhibitors; and (C) Proposed dual DNMT and HDAC inhibitors.
cLogP was calculated using DataWarrior prediction.
2