Synthesis and DNA cleavage activity of 2-hydrazinyl-1,4,5,6-tetrahydropyrimidine containing hydroxy group

Article abstract of DOI:10.1016/j.bmc.2009.05.044

2-Hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol dihydrochloride 2, as well as 2-hydrazinyl-4,5-dihydro-1H-imidazole dihydrochloride 1, was synthesized as metal-free DNA cleaving agent. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of 1 and 2. DNA cleavage efficiency of 2 exhibits remarkable increases compared with its corresponding non-hydroxy compound 1. Kinetic data of DNA cleavage promoted by 2 fit to the Michaelis-Menten-type equation with k_max of 0.0378 ± 0.0013 h^-1 giving 10⁶-fold rate acceleration over uncatalyzed DNA. The acceleration is driven by the spatial proximity of the nucleophilic hydroxy group and the electrophilic activation for the phosphodiester by the ammonium and/or guanidinium groups. In vitro cytotoxic activities toward Hela cells and human leukemia HL-60 cells were also examined, and 2 exhibits stronger cytotoxic activities than 1.

Full text of DOI:10.1016/j.bmc.2009.05.044

Bioorganic & Medicinal Chemistry 17 (2009) 4274–4279
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and DNA cleavage activity of 2-hydrazinyl-1,4,5,6-tetrahydro-
pyrimidine containing hydroxy group
a
a
a
b
c
a
a,*
Ying Shao , Yong Ding , Zhao-Li Jia , Xiao-Min Lu , Zhi-Hong Ke , Wei-Hong Xu , Guo-Yuan Lu
a
Department of Chemistry, State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, PR China
Department of Critical Care Medicine, Nanjing Zhong-Da Hospital, Southeast University School of Medicine, Nanjing 210009, PR China
Institute of Theoretical and Computational Chemistry, Laboratory of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University,
b
c
Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol dihydrochloride 2, as well as 2-hydrazinyl-4,5-dihydro-
H-imidazole dihydrochloride 1, was synthesized as metal-free DNA cleaving agent. Agarose gel electro-
Received 18 March 2009
Revised 15 May 2009
Accepted 16 May 2009
Available online 23 May 2009
1
phoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of 1 and 2. DNA
cleavage efficiency of 2 exhibits remarkable increases compared with its corresponding non-hydroxy
compound 1. Kinetic data of DNA cleavage promoted by 2 fit to the Michaelis–Menten-type equation
ꢀ1
6
with kmax of 0.0378 ± 0.0013 h giving 10 -fold rate acceleration over uncatalyzed DNA. The acceleration
is driven by the spatial proximity of the nucleophilic hydroxy group and the electrophilic activation for
the phosphodiester by the ammonium and/or guanidinium groups. In vitro cytotoxic activities toward
Hela cells and human leukemia HL-60 cells were also examined, and 2 exhibits stronger cytotoxic activ-
ities than 1.
Keywords:
Metal-free DNA cleavage
DNA cleavage
Guanidinium
Agarose gel electrophoresis
Anti-tumor activity
Ó 2009 Published by Elsevier Ltd.
1
. Introduction
It is known that positive charge group (guanidinium or ammo-
nium) is the key functionality at the active site in natural nuclease,
1
0
The cleaving agents of nucleic acid have attracted extensive
for example, staphylococcal nuclease (SNase) and bovine pancre-
1
1
attention due to their potential applications in the fields of molec-
ular biological technology and drug development.1 Metal com-
plexes as cleaving agents of nucleic acids have been widely
investigated and found to be quite efficient,2 but their pharmic
use is hampered by concerns over the lability and toxicity due to
the free radical generation during the redox processes of some
atic ribonuclease (RNase A) for nucleic acid hydrolysis, because of
the binding ability to negative groups through hydrogen bonding
1
2
and electrostatic interaction in biological molecules just as the
12a,13
phosphate of DNA or RNA.
Some compounds with positive
charge groups (ammonium or guanidinium) as nuclease mimics
for cleavage of phosphodiester have been reported, a few of them
3
3a,4b,c
transition metal, such as Cu. Recently, some small organic mole-
cules, for example, guanidinium derivatives, cyclodextrin deriva-
were identified as efficient cleavers of RNA.
Furthermore, as
4
ammonium or guanidinium group is positively charged, the corre-
sponding compounds might also be with a reasonable aqueous sol-
ubility which could be helpful for cell infiltration at physiological
conditions.
5
6
tives, macrocyclic polyamines, pseudorotaxane composed of
cucurbituril and dipeptides8 were successfully used to cleave
phosphodiester. Thus, the idea of metal-free cleavage agents are
put forward by Göbel and co-workers,4 they are considered safer
for their hydrolytic pathway of cleaving the P–O bond of phospho-
diester and have shown clinical potential. However, the studies of
7
a–c
Moreover, Cech and his co-workers reported that ribonucleic
acids are able to catalyze transesterification of their own phospho-
0
diester bonds, because of the presence of the 2 -OH group at the ri-
1
4
metal-free cleavage agents were mainly applied to active phospho-
bose of RNA as a highly efficient internal nucleophile. Very soon
it was shown that the catalysis is not restricted to intrastrand
transesterifications, but the action of the catalytic domains may
diesters, for example, nucleic acid mimic and RNA.4
a–c
As well
known, the phosphodiester bonds of DNA are extremely stable un-
der uncatalyzed physiological conditions and the half life of DNA
for hydrolysis is estimated to be about 200 million years. Thus,
1
5
also be targeted toward another RNA molecule.
9
In 1996, Göbel’s group has demonstrated that the cleavage rate
of P–O of cyclophosphate is considerably increased by introduce
the hydroxy group to their bis(guanidinium) compound though
the field of metal-free DNA cleavage agent is very challenging.
4
a
phosphoryl transfer reactions. Recently, Our group reported the
design and synthesis of the triaza crown ester derivative 1-(2-gua-
nidinoethyl)-4-(2-hydroxyethyl)-1,4,7-triazacyclononane hydro-
*
Corresponding author. Tel.: +86 25 83685613; fax: +86 25 83317761.
E-mail address: lugyuan@nju.edu.cn (G.-Y. Lu).
0
968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.05.044

Y. Shao et al. / Bioorg. Med. Chem. 17 (2009) 4274–4279
4275
chloride,13j including guanidinium serving to recognize, bind and
electrophilically activate the anionic phosphodiester through
hydrogen bonding and electrostatic interaction, and hydroxy
groups working as a nucleophilic group in the intermolecular
transphosphorylation which has been found to be efficient me-
tal-free DNA cleaving reagent. To further studying the bifunctional
cooperative effect of positive charge groups (guanidinium and/or
ammonium) and adjacent hydroxy groups on DNA cleavage, we re-
port here, the design and synthesis of 2-hydrazinyl-1,4,5,6-tetra-
assays. It is easy to understand that 2 has higher DNA cleavage
activity at acidic conditions due to its positive charge, advanta-
geous to interact with negatively charged DNA, while as pH value
increased from 6.75 to 8.0, the activity of 2 also increased might
due to the increasing tendency of proton dissociation of hydroxy
group favoring the nucleophilicity of hydroxy group.
2.2.2. Ionic strength dependence of DNA cleavage promoted
by 2
hydropyrimidin-5-ol dihydrochloride
studies of its cleavage and cytotoxic activities. In the compound
, six-membered heterocycle containing guanidinium is of enve-
2
(Scheme 1) and the
As shown in Figure 3, the DNA cleavage reactivity of the com-
pound 2 decreased along with an increase of the ionic strength,
which suggests that the positively charged compound 2 binds with
the phosphate of DNA, rather than with the DNA bases, via electro-
2
lope conformation and the appending hydroxy and ammonium
groups can bend to the same direction. As comparison, the DNA
cleavage and cytotoxic activities of 2-hydrazinyl-4,5-dihydro-1H-
imidazole dihydrochloride 1 without hydroxy group (Scheme 1),
which is the synthetic intermediate of bisantrene hydrochloride
2
2
static interactions.
2.2.3. Kinetic assays
Kinetics of pUC 19 DNA degradation has been studied. Figure 4
shows the supercoiled plasmid DNA cleavage into nicked form
with further reaction, promoted by 2 (0.463 mM) in Tris–HCl buf-
fer (pH 6.0) at 37 °C. The time course plot indicates that the exten-
sion of supercoiled DNA cleavage varies exponentially with the
reaction time giving a pseudo-first-order kinetics with an apparent
(
a commercial anti-tumor drug),16 was also studied.
2
2
. Results and discussion
.1. General aspects
ꢀ1
initial first-order rate constant (kobs) of 0.0196 ± 0.0009 h . Then,
the apparent initial first-order rate constants of DNA cleavage reac-
tions promoted by a series of various concentrations of 1 and 2 un-
der the similar conditions as described above are summarized in
Table S1 (Supplementary data). Data of 2 were fit to a curve based
on the Michaelis–Menten-type equation (kobs = kmax[enzyme]/
The synthesis of the target compounds 1 and 2 (Scheme 2) were
both achieved via a four-step reaction sequence of cyclization,
addition–elimination, nucleophilic substitution and acidification,
starting from 1,2-diaminoethane 1a and 1,3-diaminopropan-2-ol
1
7
18
16,19
20
18
2
a, respectively. The compounds 1a, 1b, 1c,
2a, 2b,
1
9
(K
d
+ [enzyme])), where kobs is the apparent first-order rate con-
stant at various concentrations, the kmax is the maximum rate con-
stant at saturation concentration and is the apparent
and 2c were synthesized according to corresponding literatures.
1
13
The compounds 1 and 2 were characterized by H NMR, C NMR,
ESI-MS spectra (Figs. S1–S10, Supplementary data), and elemental
analysis.
K
d
23
dissociation constant for the enzyme-substrate compound. The
kinetics profiles of the supercoiled DNA cleavage at various con-
centrations of 1 and 2 are shown in Figure 5. The kinetic data are
linearized through a double-reciprocal plot of 1/kobs versus 1/[en-
zyme] ([enzyme] is the concentrations of 2), giving the first-order
2
2
.2. DNA cleavage activity
.2.1. pH dependence of DNA cleavage promoted by 1 and 2
ꢀ1
rate constant kmax of 0.0378 ± 0.0013 h and the apparent dissoci-
ation constant K of 0.425 mM (Fig. S11, Supplementary data). The
Cleavage reactions of supercoiled pUC 19 DNA (0.05 mM bp)
were carried out at 37 °C in 50 mM Tris–HCl buffer with a total vol-
ume of 15 L. The results of cleavage reactions were monitored by
d
non-hydroxy-substitutive 1 shows very weak cleavage activity (
Fig. 5), which indicates that the pendant hydroxy group effectually
increases the DNA cleavage activity.
l
agarose gel electrophoresis (80 V for 1.5 h). Bands were visualized
TM
by UV light and photographed using a DigiDoc–It gel imaging and
documentation system (version 1.1.23, UVP, Inc., Unpland, CA). the
intensity of the DNA bands was estimated by TotalLab image anal-
ysis software (version 2.01), and the supercoiled plasmid DNA val-
ues were corrected by a factor of 1.3 on the basis of average
2
.3. Studies on mechanism of DNA cleavage
Although no transition metal ion or any other functional group
2
1
of 2 can generate reactive oxygen species (ROS), when particularly
with the employment of DNA derived from biological sources, the
presence of ROS is possible. To verify if ROS are, at least in part,
responsible for the cleavage of DNA promoted by 2, reactions were
carried out in the presence of typical scavengers for singlet oxy-
gen (NaN , 10 mM), for superoxide (KI, 10 mM), and for hydroxyl
radical (1 mM DMSO and 1 mM t-BuOH). Figure 6 shows that there
is no evident inhibition effect on the DNA cleavage in the presence
3
of all scavengers (NaN , DMSO, t-BuOH and KI), which rules out the
literature estimates of lowered binding of EB to this structure.
Figure 1a and b displays the results of agarose gel electrophoresis
of pH-dependence assays which indicates that the supercoiled
DNA (form I) relaxes to form a nicked circular DNA (form II) in
the presence of 1 and 2 when the pH is in the range of 6.0–8.5.
Figure 2 presents the pH-dependence profile which indicates that
the DNA cleavage ability of 2 is stronger than that of 1, and the
optimal pH for DNA cleavage in the presence of 2 is pH 6.0. There-
fore, pH 6.0 was chosen for following agarose gel electrophoresis
2
4
3
involvement of these reactive oxygen species, at least in a free and
diffusible form. Therefore, DNA cleavage promoted by 2 might
mainly through the non-oxidative pathway.
According to Göbel’s report4a, their synthesized bis(guanidi-
nium) compound with hydroxy group can cleave P–O bonds of
OH
. 2HCl
cyclophosphate though phosphoryl transfer reactions. Further-
HN
HN
N
.
2
HCl
HN
HN
N
13j
more recently our group
has reported that 1,4,7-triazacyclo-
nonane equipped with guanidinium and hydroxy group can
effectually cleavage P–O bonds of bis(2,4-dinitrophenyl) phosphate
NH
2
NH2
(
BDNPP) and DNA via transphosphorylation reaction. Hereby, it is
1
2
very possible that the phosphodiester bond of DNA would be also
Scheme 1. The structure of the compounds 1 and 2.
cleaved by 2 via phosphoryl transfer reactions. In order to simplify

4
276
Y. Shao et al. / Bioorg. Med. Chem. 17 (2009) 4274–4279
H
N
H
N
H
N
NH2
NH2
(
a)
(b)
(c)
S
SCH3
HCl
NHNH2
2HCl
N
H
N
N
1
a
1b
1
c
1
OH
OH
OH
OH
(
d)
(e)
(f)
. 2HCl
HN
NH
N
NH
HN
HN
N
NH2 NH2
HCl
S
SCH3
NH2
2
a
2b
2c
2
Scheme 2. Synthesis of the compounds 1 and 2. Reagents and conditions: (a) CS
then reflux, 24 h, 81.0%; (c) ice bath, N , H NNH
ꢁH
0 min, 43.6%; (e) concd HCl, CH OH, then reflux, 24 h, 65.3%; (f) ice bath, N
2
, EtOH/H
2
O, 60 °C, 2 h; then 100 °C, 1 h; concd HCl, rt, 10 h, 76.5%; (b) concd HCl, CH
3
OH,
2
2
2
2
O, EtOH, rt, 1 h, then reflux, 4 h; 1 M HCl, rt, 88.4%; (d) CS
2
, EtOH/H O, rt, 1 h; microwave 180 w, 20 min, concd HCl,
2
1
3
2
, H
2
NNH
2
ꢁH
2
O, EtOH, rt, 0.5 h, then reflux, 4 h; 1 M HCl, rt, 78.9%.
1
2
3
4
5
6
7
8
80
a
b
1
2 3 4 5 6 7
Form II
Form I
II
I
60
1
2
3
4
5
6
7
8
9
Form II
Form I
40
20
Figure 1. (a) Agarose gel electrophoresis (1%) of pUC 19 DNA (0.05 mM bp)
incubated for 24 h at 37 °C with 1.10 mM 1 in different pH buffer. Lanes 1–8, DNA
control, pH 6.0, 6.5, 7.0, 7.25, 7.5, 8.0 and 8.5, respectively. (b) Agarose gel
electrophoresis (1%) of pUC 19 DNA (0.05 mM bp) incubated for 24 h at 37 °C with
0
10 20 30 40 50 60
Concentration (NaCl, mM)
1
.10 mM 2 in different pH buffer. Lanes 1–9, DNA control, pH 6.0, 6.5, 6.75, 7.0, 7.25,
7
.5, 8.0 and 8.5, respectively.
Figure 3. Ionic strength dependence of the plasmid DNA cleavage promoted by 2
0. 70 mM). The reactions were carried out at 37 °C for 24 h in 50 mM Tris–HCl
(
buffer (pH 6.0). Ionic strength was controlled by NaCl. Inset of Figure 3 shows the
results of the agarose gel electrophoresis (1%) of the ionic strength variable reaction
products. Lanes 1–7, ionic strength of control, 0, 10.0 mM, 20.0 mM, 37.5 mM,
100
5
0.0 mM and 62.5 mM NaCl, respectively.
8
6
4
2
0
0
0
0
0
2
0
.0
0.1
-0.2
0.3
-0.4
0.5
-0.6
0.7
1 2 3 4 5 6 7 8 9
-
II
I
1
-
6.0
6.5
7.0
pH
7.5
8.0
-
Figure 2. pH dependent profile for DNA cleavage promoted incubated for 24 h by
.10 mM 1 ( ) and 1.10 mM 2 (N) in different pH buffer (50 mM Tris–HCl) at 37 °C.
1
-
4
8
12 16 20 24 28 32
Time (hour)
the investigation of the mechanism, simple DNA mimic (BDNPP)
was taken into account. If the transphosphorylation pathway is
possible, when in the process of cleavage of BDNPP, the intermedi-
ate I (displayed in Scheme 3) should possibly generated.
Figure 4. Time course of pUC19 DNA (0.05 mM bp) cleavage promoted by
2
(
0.463 mM) in 50 mM Tris–HCl buffer (pH 6.0) at 37 °C. Inset of Figure 4 is the
results of the agarose gel (1%) electrophoresis of the time-variable reaction
products. Lanes 1–9, reaction time of 0 h, 4.95 h, 9.07 h, 13.07 h, 23.12 h, 25.35 h,
In order to investigate the possibility of stable existence of as-
27.33 h, 29.33 h and 31.48 h, respectively.
sumed intermediate I, theoretical calculations were applied. Figure
displays the calculated structure of the intermediate I.2 As ex-
5
7
pected, the six-membered heterocycle of compound 2 is of enve-
lope conformation, all the atoms of guanidinium group are
coplanar, and the appending hydroxy and ammonium groups can
bend to the same direction. A strong intramolecular hydrogen
bond is formed between the phosphoryl and ammonium groups
is one of the possible mechanisms for the DNA cleavage promoted
by 2. Therefore, it can explain the stronger cleavage activity of 2
than that of 1 without hydroxy group.
2.4. Cytotoxic activities
(
the corresponding Oꢁ ꢁ ꢁH distance is 1.67 Å and the N–Hꢁ ꢁ ꢁO angle
is 165°). As the intermediate I can stably exist, we assume that the
phosphodiester bond of BDNPP could be cleaved by 2 via a trans-
phosphorylation pathway. Similar to BDNPP, transphosphorylation
To evaluate the anti-tumor activity, Hela and Human leuke-
mia HL-60 cells were exposed to different concentrations of
the compounds 1 and 2 for 72 h. Cell viability was determined

Y. Shao et al. / Bioorg. Med. Chem. 17 (2009) 4274–4279
4277
0.020
0.015
0.010
0.005
0.000
2
1
0.0
0.1
0.2
0.3
0.4
0.5
Concentration, mM
Figure 5. Kinetics plot of kobs versus various concentrations of 1 ( ) and 2 (j).
%
%
Cleaved supercoiled DNA
supercoiled DNA
a 1
00
80
60
40
20
0
1
2
3
4
5
6
DNA No
NaN3 KI t-BuOH DMSO
Control inhibitor
Figure 7. The optimized geometry of the intermediate I. The intramolecular
hydrogen bond is represented as dashed line.
b
1
2
3
4
5
6
Form II
Form I
Figure 6. (a) Histogram representing of pUC19 plasmid DNA (0.05 mM bp) cleaved
by 2 (0.7 mM) in the presence of standard radical scavengers incubated for 24 h at
120
100
3
7 °C in pH 6.0 buffer (50 mM Tris–HCl). (b) Agarose gel electrophoresis of radical
scavengers inhibition reactions. Lanes 1–6, DNA control, DNA + 2, DNA + 2 + NaN
3
8
6
4
2
0
0
0
0
0
(
(
10 mM), DNA + 2 + KI (10 mM), DNA + 2 + t-BuOH (1 mM), and DNA + 2 + DMSO
1 mM), respectively.
1
2
HL-60 Cell
O N
2
O
O
P
-5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0
O N
O
2
H
O
NH2
NH
log(C), C = [compound], M
H
N
Figure 8. Inhibitory effect of 1 and 2 on the proliferation of HL-60. Cells were
exposed to the compounds for 72 h. The relative survival rate was determined in
relation to that of untreated control cells, which was set as 100%. Data were the
mean ± SD of three experiments and each experiment included triplicate wells.
NH
intermediate I
Scheme 3. The structure of the possible resultant intermediate I of BDNPP treated
with 2.
3. Conclusions
In conclusion, a 2-hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol
dihydrochloride 2 as DNA cleaving reagent was designed and pre-
pared. pH and ionic strength dependences of the DNA cleavage
promoted by 1 and 2 were studied by agarose gel electrophoresis.
Kinetic data of DNA cleavage promoted by 2 fit to the Michaelis–
by MTT assays.26 The inhibition percentages were listed in Table
S2 (Supplementary data). According to these results of the cyto-
toxic assays, we conclude that compound 2 exhibit stronger
cytotoxic effects than 1, and HL-60 cells are proved to be more
sensitive to these compounds than Hela cells. In order to further
evaluate the anti-tumor activity of the compounds 1 and 2, IC50
values against HL-60 cells were calculated as shown in Figure 8.
The IC50 values are 6.91 ꢂ 10 M for 1 and 9.20 ꢂ 10 M for 2,
respectively. According to these results of the cytotoxic assays,
we conclude that 2 exhibits stronger cytotoxic effects than 1,
which is consistent with the order of the DNA cleavage abilities
of these compounds.
ꢀ
1
Menten-type equation with kmax of 0.0378 ± 0.0013 h giving an
6
ꢃ10 -fold rate acceleration over uncatalyzed DNA. DNA cleavage
efficiency of 2 exhibits remarkable increases compared with its
corresponding non-hydroxy-side-armed compound 1, and the the-
oretical calculation result shows that the acceleration of the DNA
cleavage promoted by 2 is most possibly due to the cooperative ef-
fect of the positive charge center and hydroxy groups. Further-
more, a preliminary study of the antitumor activity shows that 2
ꢀ4
ꢀ5

4
278
Y. Shao et al. / Bioorg. Med. Chem. 17 (2009) 4274–4279
exhibits stronger cytotoxic effects than 1. The IC50 value against
HL-60 cells of 2 is 9.20 ꢂ 10 M.
1 (1.53 g, 8.84 mmol) was obtained after dryness under vacuum.
ꢀ5
1
13
Yield: 88.4%. H NMR (300 MHz, D
NMR (75 MHz, D O) d 42.73 (CH
MꢀH ꢀ2Cl ] , calcd 101.08, found 101.01. Anal. Calcd for
C H Cl N : C, 20.82; H, 5.82; N, 32.38. Found: C, 20.82; H, 5.81;
2
O) d 3.55 (s, 4H, 2 ꢂ CH
2
).
C
2
2
), 159.87 (N C). ESI-MS m/z
3
+
ꢀ +
[
4
4
. Experimental
3
10
2
4
N, 32.27.
.1. Materials
4
.3.4. 5-Hydroxytetrahydropyrimidine-2(1H)-thione (2b)
Compound 2b was prepared according to the literature method,
All reagents, purchased from commercial sources, were of ana-
lytical grade and used without further purification. pUC 19 plasmid
DNA was purchased from TaKaRa Biotechnology Co. Ltd, and the
purity was checked by agarose gel electrophoresis; concentrations
were determined by UV spectroscopy using the extinction coeffi-
20
1
off-white powder was obtained. Yield: 43.6%. H NMR (300 MHz,
DMSO-d ) d 2.90–2.96 (m, 2H, NCH ), 3.14–3.19 (m, 2H, NCH ),
.90 (br s, 1H, OCH), 5.19 (br s, 1H, OH), 7.74 (br s, 2H, 2 ꢂ NH).
6
2
2
3
cient appropriate for double-stranded DNA (1.0 OD260 = 50 lg/
4
.3.5. 2-(Methylthio)-1,4,5,6-tetrahydropyrimidin-5-ol
mL). Agarose was from Oxoid Limited (Basingstoke, UK). Ethidium
bromide (EB) was from Amresco. Inc. Tris(hydroxymethyl)amino-
methane (Tris–Base) was from Robiot Co. Ltd. Bromophenol blue,
glycerol and ethylenediaminetetraacetic acid (EDTA) were com-
mercially available. Deionized water was obtained from an ion-ex-
change column from doubly distilled water. All solvents were
purified by the standard procedure. All aqueous solutions were
prepared from deionized water. Loading buffer IV was prepared
hydrochloride (2c)
Compound 2c was prepared as above-described synthetic
method, white powder was obtained.18 Yield: 65.3%. 1H NMR
(
300 MHz, D
2
O) d 2.48 (s, 3H, SCH
3 2
), 3.33–3.38 (m, 2H, NCH ),
3
.44–3.49 (m, 2H, NCH ), 4.35 (br s, 1H, OCH).
2
4
.3.6. 2-Hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol
2
7
dihydrochloride (2)
As above-described synthetic method, white powder 2 was ob-
according to literature (the final solution contained 0.25% bromo-
phenol blue, 40% saccharose and 500 M of EDTA was stored under
l
1
9
1
tained after dryness under vacuum.
300 MHz, D O) d 3.20–3.25(m, 2H, 2NCH
NCH ), 4.21(br s, 1H, OCH). C NMR (75 MHz, D
CH ), 58.42 (HOCH), 153.35 (N
31.09, found 131.00. Anal. Calcd for C
Yield: 78.9%. H NMR
), 3.30–3.35(m, 2H,
O) d 43.63
4
°C).
(
2
2
1
3
2
2
2
+
- +
(
2
3
C). ESI-MS m/z [M-H -2Cl ] , calcd
O: C, 23.66; H,
4
.2. Apparatus
1
4 2 4
H12Cl N
1H NMR and 13C NMR spectra were recorded on a Brucker AM
5.96; N, 27.59. Found: C, 23.64; H, 5.92; N, 27.35.
3
00 spectrometer. Mass spectra were obtained on an electro-
TM
spray mass spectrometer (LCQ , Finnigan) in positive mode.
Elemental analyses were performed on a Perkin Elmer 240C
apparatus. The pH value was confirmed with an ORION868 pH
meter with an Ag/AgCl electrode as the reference electrode in
a saturated KCl solution at room temperature. Agarose gel elec-
trophoresis was conducted with a DYY–5 electrophoresis appara-
tus. Bands were visualized by UV light and photographed using a
4.4. Agarose gel electrophoresis assays
The plasmid DNA cleavage experiments were performed using
pUC19 DNA in Tris–HCl buffer. Reactions were performed by incu-
bating DNA (0.05 mM bp) at 37 °C in the dark in the presence/ab-
sence of the compound 1 or 2 for the indicated time. All
reactions were quenched by loading buffer (3–3.5
gel electrophoresis was carried out on a 1% agarose gel in 0.5 ꢂ TAE
(Tris-acetate–EDTA) buffer containing 0.5 g /mL EB at 80 V for
lL). Agarose
TM
DigiDoc–It gel imaging and documentation system (version
1
.1.23, UVP, Inc., Unpland, CA). The intensity of the DNA bands
l
was estimated by TotalLab image analysis software (version
1.5 h. The resolved bands were visualized with a UV transillumina-
2
4
4
.01).
tor and quantified using TotalLab 2.01 software.
.3. General procedure for the synthesis of compounds
4
.5. Computational details
.3.1. Imidazolidine-2-thione (1b)
Compound 1b was prepared according to the literature method,
Theoretical calculations were applied to investigate the stability
of the intermediate I. The polarized continuum model (PCM)28 was
employed to include the solvent effect. The optimized structure for
the intermediate I was further confirmed by the frequency calcula-
white powder was obtained.17 Yield: 76.5%. 1H NMR (300 MHz,
CDCl
3
) d 3.77 (s, 4H, 2 ꢂ NCH
2
).
2
9
tions. The B3LYP exchange-correlation functional and the stan-
dard 6-31G(d) basis set were used. All the calculations were
4
.3.2. 2-(Methylthio)-4,5-dihydro-1H-imidazole hydrochloride
3
0
(
1c)
Compound 1c was prepared according to the literature method,
white powder was obtained.18 Yield: 81.0%. 1H NMR (300 MHz,
O) d 2.41 (s, 3H, SCH ), 3.72 (s, 4H, NCH ).
performed with GAUSSIAN03 program.
.6. Cytotoxic activity studies
Tumor cells were treated with different compounds for 72 h and
4
D
2
3
2
4
.3.3. 2-Hydrazinyl-4,5-dihydro-1H-imidazole dihydrochloride
incubated with 4 mg/mL MTT for 4 h. The amount of MTT formazan
produced was determined by measuring absorbance at 540 nm.
1
6,19
(
1)
A solution of 1c (1.53 g, 10.0 mmol) in ethanol (10 mL) was
cooled to below 5 °C, and 85% hydrazine hydrate (0.65 mL,
1.4 mmol) was added in one portion under the protection of
1
Acknowledgements
nitrogen. The resulting mixture was stirred for 1 h and then heated
to reflux for 4 h. The solution was evaporated under vacuum fol-
lowed by azeotropy with water (3 ꢂ 20 mL) to remove the super-
fluous hydrazine hydrate. The residue was acidified with 10 mL
of 1 M HCl and the solvent was removed in vacuum. White powder
This work was supported by the National Basic Research (Grant.
No. 2007CB925103) and National Science Foundation (Grant. No.
20872061) of China. We acknowledge gratefully Mr. Li-Qing Qiang
for the assistance with the electrophoresis assays.

Y. Shao et al. / Bioorg. Med. Chem. 17 (2009) 4274–4279
4279
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Supplementary data
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