Synthesis and antitumor activity evaluation of a novel combi-nitrosourea prodrug: Designed to release a DNA cross-linking agent and an inhibitor of O6-alkylguanine-DNA alkyltransferase

Article abstract of DOI:10.1016/j.bmc.2016.03.041

The drug resistance of CENUs induced by O⁶-alkylguanine-DNA alkyltransferase (AGT), which repairs the O⁶-alkylated guanine and subsequently inhibits the formation of dG-dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer⁺ glioma cells compared with ACNU, BCNU, and their respective combinations with O⁶-benzylguanine (O⁶-BG). Quantifications of dG-dC cross-links induced by 6 were performed using HPLC-ESI-MS/MS. Higher levels of dG-dC cross-link were observed in 6-treated human glioma SF763 cells (mer⁺), whereas lower levels of dG-dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O⁶-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O⁶-BG.

Full text of DOI:10.1016/j.bmc.2016.03.041

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumor activity evaluation of a novel
combi-nitrosourea prodrug: Designed to release a DNA cross-linking
agent and an inhibitor of O6-alkylguanine-DNA alkyltransferase
⇑
Guohui Sun, Na Zhang, Lijiao Zhao , Tengjiao Fan, Shufen Zhang, Rugang Zhong
Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China
a r t i c l e i n f o
a b s t r a c t
The drug resistance of CENUs induced by O6-alkylguanine-DNA alkyltransferase (AGT), which repairs the
O6-alkylated guanine and subsequently inhibits the formation of dG–dC cross-links, hinders the applica-
tion of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a
promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a
new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-
1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized
and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The
results indicated that 6 exhibited higher cytotoxicity against mer+ glioma cells compared with ACNU,
BCNU, and their respective combinations with O6-benzylguanine (O6-BG). Quantifications of dG–dC
cross-links induced by 6 were performed using HPLC–ESI-MS/MS. Higher levels of dG–dC cross-link were
observed in 6-treated human glioma SF763 cells (mer+), whereas lower levels of dG–dC cross-link were
observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU.
The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specif-
ically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds
were formed between the O6-BG analogs released from 6 and the five residues in the active pocket of
AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O6-BG.
Ó 2016 Elsevier Ltd. All rights reserved.
Article history:
Received 5 February 2016
Revised 22 March 2016
Accepted 25 March 2016
Available online xxxx
Keywords:
Combi-nitrosourea
O6-alkylguanine-DNA alkyltransferase
dG–dC cross-links
Drug resistance
Cytotoxicity
1. Introduction
(ACNU), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chlor-
oethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chlor-
Chloroethylnitrosoureas (CENUs), such as 1-[(4-amino-
2-methyl-5-pyrimidinyl)-methyl]-3-(2-chloroethyl)-3-nitrosourea
oethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (me-CCNU) (Fig. 1),
are an important type of bifunctional alkylating agents widely used
for the clinical treatment of cancer.1–4 They are highly effective for
the treatment of brain tumors due to their ability to penetrate the
blood–brain barrier.1,2 Chloroethylation of the O6-position in DNA
guanine residues is the major source of the antitumor activity of
CENUs because the O6-chloroethylating adducts can result in the
formation of dG–dC interstrand cross-links (ICLs), which can inhibit
DNA double-strand separation during replication and transcription,
leading to cell apoptosis if not repaired correctly.5–9 As shown in
Figure 2, CENUs can decompose under physiological conditions to
produce a chloroethylating intermediate that attacks the deoxygua-
nosine (dGuo) to form O6-(2-chloroethyl)deoxyguanosine
(O6-ClEtdGuo). Then O6-ClEtdGuo undergoes intramolecular
cyclization to yield N1,O6-ethanodeoxyguanosine (N1,O6-EtdGuo)
Abbreviations: CENUs, chloroethylnitrosoureas; ACNU, 1-[(4-amino-2-methyl-
5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea; BCNU, 1,3-bis(2-chlor-
oethyl)-1-nitrosourea; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; me-CCNU,
1-(2-chloroethyl)-3-(4-methyl-cyclohexyl)-1-nitrosourea; ICLs, interstrand cross-links;
O6-ClEtdGuo, O6-(2-chloroethyl)deoxyguanosine; N1,O6-EtdGuo, N1,O6-ethanodeoxy-
guanosine; dGuo, deoxyguanosine; dCyd, deoxycytidine; AGT, O6-alkylguanine-DNA
alkyltransferase; Cys145, cysteine145; O6-BG, O6-benzylguanine; HPLC–ESI-MS/MS,
high-performance liquid chromatography electrospray ionization tandem mass spec-
trometry; DMF, N,N-dimethylformamide; PBS, phosphate buffer saline; MEM-EBSS,
minimum essential medium with Earle’s balanced salts; FBS, fetal bovine serum; t1/2
,
half-life; CT DNA, calf thymus DNA; PDB, Protein Data Bank; O6-MG, O6-methylgua-
nine; RMSD, root mean square deviation; TLC, thin-layer chromatography; Hz, Hertz;
HRMS, high-resolution mass spectrum; TEA, triethylamine; SD, standard deviation;
CCK8, Cell Counting Kit-8; DMSO, dimethyl sulfoxide; DNase I, deoxyribonuclease I;
CIAP, alkaline phosphatase; SRM, selected reaction monitoring.
followed by
a
second alkylation of the complementary
deoxycytidine (dCyd) to produce an ethyl bridge between the N1
position of guanine and the N3 position of cytosine, resulting in
the formation of a dG–dC cross-link.1,2
⇑
Corresponding author. Tel.: +86 10 67391667.
E-mail address: zhaolijiao@bjut.edu.cn (L. Zhao).
http://dx.doi.org/10.1016/j.bmc.2016.03.041
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
N
O
N
N
H
N
NH
N
N
N
Cl
Cl
CH2
H2N
Cl
CH3
O
O
O
ACNU
BCNU
N
N
R
N
Cl
H
O
O
O
CENUs
N
N
N
N
H
N
H
N
Cl
Cl
O
O
CH3
CCNU
me-CCNU
Figure 1. Typical CENUs used in the clinical treatment of cancer.
O
RN
C
O
N
N
R
N
N
N
OH
Cl
Cl
H
O
CENUs
O
N
OH-
NH
N
N
CH2CH2
NH2
N
Anticancer
N
N
N
O
N
Cl
DNA
DNA
dG-dC cross-link
dGuo
N2
dCyd
O
S
N
Cl
AGT
N
N
O
O
N
Cl-
N
N
N
N
N
N
NH2
AGT
AGT
dGuo
Cl
AGT
S
DNA
N
H2N
N
NH2
ClEt-AGT
AGT-DNA crosslink
DNA
N1, O6-EtdGuo
DNA
O6-ClEtdGuo
Drug resistance
Drug resistance
Figure 2. The general mechanisms for the formation and repair of dG–dC cross-links.
However, O6-alkylguanine-DNA alkyltransferase (AGT) in tumor
pseudosubstrate to form S-benzylcysteine at the active site of
AGT to inactivate the protein, is currently undergoing clinical trials
as an adjuvant in combination with BCNU.31–34 Although O6-BG is a
promising AGT inhibitor, its modest inactivating ability and poor
water solubility limit its clinical effectiveness.15,27,29,30,33,34 The
concept of using a ‘combi-molecule’ to overcome the drug resis-
tance has been attempted. Qiu et al.35,36 synthesized a series of
combi-nitrosoureas with epidermal growth factor receptor tyro-
sine kinase inhibitory properties while maintaining DNA cross-
linking reactivity.
In this study, we describe the synthesis, antitumor activity and
mechanism of action of a new combi-nitrosourea prodrug 3-(3-
(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-
1-nitrosourea (6), which is designed to release a DNA cross-linking
agent and an inhibitor of AGT (Scheme 1). This study is expected
to provide a foundation for the development of novel CENU
chemotherapies with high efficacy.
cells can repair the intermediate O6-ClEtdGuo and N1,O6-EtdGuo
by transferring the O6-position lesions to its own active site cys-
teine145 (Cys145) residue, thereby inhibiting the generation of
lethal dG–dC cross-links and leading to drug resistance
(Fig. 2).10–15 After accepting the alkyl groups, AGT is rapidly
degraded by ubiquitination-dependent proteolysis.16,17 Based on
the AGT levels, most human cell lines can be divided into two
classes: cells with low or no AGT activity (termed merꢀ), which
are sensitive to the cytotoxic effects of CENUs and were observed
to have high levels of ICLs, and cells with high levels of AGT
(termed mer+), which are resistant to these effects and exhibited
low levels of ICLs.5,18–22 Because high levels of AGT can cause a
strong resistance to CENU chemotherapy in tumor cells, a series
of AGT inhibitors were synthesized and were used as adjuvants
to improve the chemotherapeutic effects of CENUs.23–30 The
potent inactivator O6-benzylguanine (O6-BG), which acts as a
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G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Cl
N
N
OH
Induction of dG-dC cross-links
O
Cl
N
O
9
NH
N
N
C O
NH2
O
N
N
N
O
O
N
N
H
NH2
H2O
CO
N
N
N
6
-
N
H
2
N
H
N
NH2
N
NH2
8
4
Inactivation of AGT
Inactivation of AGT
Scheme 1. The decomposition pathway of combi-chloroethylnitrosourea prodrug 6.
benzyl)-2,2,2-trifluoroacetamide (3) in the presence of potassium
t-butoxide. The trifluoroacetyl protecting group of intermediate 3
was removed by refluxing in the presence of a methanol/water
solution to give O6-[3-(aminomethyl)benzyl]guanine (4). The reac-
tion of 4 with 2-chloroethyl isocyanate in anhydrous DMF yielded
1-(3-(((2-amino-9H-purin-6-yl)oxy)-methyl)benzyl)-3-(2-chlor-
oethyl)urea (5) followed by nitrosation with nitrosonium tetraflu-
oroborate in acetonitrile containing a moderate amount of glacial
acetic acid at 0 °C to afford the desired final product 6.
2. Results and discussion
2.1. Chemistry
According to the results obtained from previously published
studies,30,37 the meta-substitution at the benzyl of O6-BG can
result in higher inhibitory activity against AGT than that of ortho-
or para-substitution. Thus, the novel combi-nitrosourea prodrug
6 was synthesized and designed to release the meta-substituted
O6-BG analog 6-((3-(isocyanatomethyl)benzyl)oxy)-9H-purin-2-
amine (8) and its hydrolysate meta-substituted O6-BG analog
O6-[3-(aminomethyl)benzyl]guanine (4) as an AGT inhibitor, along
with chloroethylating intermediate 9, which can induce dG–dC
cross-links. The synthesis of 6 is shown in Scheme 2. Briefly,
1-(2-amino-9H-purin-6-yl)-1-methylpyrrolidinium chloride (2) was
prepared by the reaction of 2-amino-6-chloroguanine (1) with N-
methylpyrrolidine in anhydrous N,N-dimethylformamide (DMF)
at room temperature. Then, 2 was condensed with 2,2,2-triflu-
oro-N-(3-(hydroxymethyl)benzyl)-acetamide (7) under argon
atmosphere to give N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)
2.2. Stability
The degradation of 6 was monitored by HPLC-UV analysis in
phosphate buffer saline (PBS) and in minimum essential medium
with Earle’s balanced salts (MEM-EBSS) supplemented with 10%
fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and
100 lg/mL streptomycin). The stability study suggested that 6
has similar decomposition properties with classical CENUs. As
shown in Figure 3, the half-lives (t1/2) of 6 in PBS and MEM-EBSS
were approximately 30 min and 18 min, respectively. The t1/2
O
O
CF3
CF3
NH
NH2
NH
Cl
N
O
HO
O
N
H3C
Cl
N
N
c
a
N
7
b
N
N
N
N
N
N
H
NH2
N
H
N
H
N
4
NH2
N
2
NH2
N
H
N
3
NH2
1
Cl
Cl
O
O
NH
N
O
NH
NH
N
O
O
e
N
N
d
N
N
N
H
N
H
NH2
N
5
NH2
N
6
Scheme 2. Synthesis of the combi-nitrosourea prodrug 6a. aReagents and conditions: (a) (1) N-methylpyrrolidine, DMF, room temperature, 18 h; (2) addition of acetone to
complete precipitation. (b) (1) Potassium t-butoxide, DMF, argon atmosphere, room temperature, 4 h; (2) addition of glacial acetic acid to neutralize excess potassium t-
butoxide. (c) Potassium carbonate, methanol/water (17:1), reflux, 2 h. (d) 2-Chloroethyl isocyanate, DMF, 0 °C to room temperature, 2 h. (e) Nitrosonium tetrafluoroborate,
acetonitrile, glacial acetic acid, 0 °C, 3 h.
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4
G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
SF763 cells, which express high levels of AGT. The results showed
that 6 (IC50 = 35 M) was significantly more cytotoxic than ACNU
(IC50 > 1000 M), BCNU (IC50 = 100 M) and their respective com-
binations with O6-BG (1000
and 55 M, respectively)
l
l
l
lM
l
(Fig. 4E and F). Considering the possible cytotoxicity of the final
species decomposed from 6, cell survival assays were performed
by treating SF126, SF767 and SF763 cells with compound 4. The
result indicated that compound 4 was nontoxic to cell growth with
a death rate below 3%.
The high sensitivity of SF767 and SF763 cells to 6 suggested that
the AGT-mediated resistance was effectively weakened by expo-
sure to 6. The high cytotoxicity of 6 against mer+ cells may be
due to the inhibition of AGT activity by the inhibitor released from
6, thereby enhancing the sensitivity of these cells to DNA cross-
linking agent 9. The chemosensitivity of cells to ACNU or BCNU lar-
gely depends on the levels of AGT, while the dependence was
mostly deprived by 6. In contrast, for SF126 cells (Fig. 4B), 6 exhib-
ited higher IC50 compared with BCNU alone and its combination
with O6-BG, which may be due to the release of the highly cyto-
toxic 2-chloroethyl isocyanate from the decomposition of BCNU
and the minimized function of 8 or 4 on low AGT-expressed
SF126 cells. The lower cytotoxicity of 6 against SF126 cells com-
pared with SF767 and SF763 cells indicated that the superiority
of 6 could be specifically reflected in mer+ cells.
2.4. Quantitation of dG–dC cross-links by HPLC–ESI-MS/MS
analysis
On the basis of the hypothesis that the dG–dC cross-links may
be used as an indicator for predicting the anticancer efficiency of
CENUs, which was put forward in our previous studies,41,42 we
quantified the dG–dC cross-links induced by 6 using HPLC–ESI-
MS/MS to explore the relationship between the levels of DNA
cross-link and the cytotoxicity of 6. The isotope dilution HPLC–
ESI-MS/MS methodology has been validated for the quantitation
of dG–dC cross-links in calf thymus DNA (CT DNA) and cells treated
with CENUs, as described in our previous study.22 The quantitative
calibration curve was constructed by plotting the selected reaction
monitoring (SRM) peak area ratio of dG–dC to 15N3-dG–dC against
the corresponding concentration ratio with a correlation coeffi-
cient (R2) of 0.9999 (Fig. S2 in the Supplementary materials). The
typical SRM ion chromatograms of dG–dC and 15N3-dG–dC internal
standard in the DNA enzymatic hydrolysates are shown in Figure 5.
The peak corresponding to dG–dC was coeluted with 15N3-dG–dC
Figure 3. The degradation of 6 performed in (A) PBS and (B) MEM-EBSS over an 8 h
incubation.
was similar to that of 15 min of BCNU in serum-containing
medium.38 The degradation of 6 was accompanied by the genera-
tion of a main peak containing 8 and a minor peak containing 4
(see Fig. S1A in the Supplementary materials). The two products
were both monitored by MS/MS with the transitions of m/z
271 ? 254 for 4 and m/z 297 ? 254 for 8 (see Fig. S1B in the Sup-
plementary materials). This is also supported by the general
decomposition pathway described previously, which indicated
that chloroethyl-diazohydroxide, isocyanate and amine generated
from hydrolysis of isocyanate were the main decomposition prod-
ucts of CENUs.1,2,38
internal standard with
a retention time of approximately
11.24 min. No signal of dG–dC cross-links was detected in the con-
trol CT DNA or in the control cell samples, suggesting no interfer-
ence or contamination in the analyte channels from the internal
standard or enzymatic hydrolysates.
2.3. Sensitivity of human glioma cells to 6
To demonstrate the efficacy of the dual functions of the combi-
chloroethylnitrosourea prodrug, 6 was tested in a cell survival
assay against three human glioma cells with different AGT activi-
ties, namely, SF126 (merꢀ, little or no AGT activity), SF767 (mer+,
61 fmol/106 cells) and SF763 (mer+, 119 fmol/106 cells).39,40 The
cytotoxic effects of 6 were compared with ACNU, BCNU and the
combination of ACNU or BCNU with O6-BG (Fig. 4). As shown in
CT DNA was treated with different concentrations of 6 (0.1, 0.2,
and 0.4 mM) for 12 h. As shown in Figure 6A (data listed in Table S1
in the Supplementary materials), the formation of dG–dC cross-
links was concentration dependent. The levels of dG–dC cross-link
in CT DNA treated with 6 were lower than those of ACNU and
BCNU over the tested concentration range. However, the levels of
dG–dC cross-link in human glioma SF763 cells (Fig. 6B, data listed
in Table S2 in the Supplementary materials) induced by 6 were sig-
nificantly higher than those of ACNU and BCNU over the same con-
centration range as described with CT DNA. These combined
results indicate that the increased levels of dG–dC cross-link in
SF763 cells after exposure to 6 were due to the inhibition of AGT
activity by O6-BG analogs 8 and its hydrolytic product 4 released
from the decomposition of 6. In contrast, the decreased levels of
dG–dC cross-link in CT DNA treated with 6 may result from the
invalidation of 8 and 4 in the absence of AGT in vitro.
Figure 4A and B, 6 (IC50 = 50
ACNU (IC50 = 320
M) and its combination with O6-BG (IC50
310 M) against SF126 cells, while it exhibited lower toxicity than
BCNU (IC50 = 20
M) alone and its combination with O6-BG
(IC50 = 25 M). For SF767 cells, which express moderate levels of
AGT, 6 (IC50 = 25 M) exhibited significantly higher cytotoxicity
than ACNU alone (IC50 = 500
M) and its combination with O6-BG
(IC50 = 400 M) (Fig. 4C), and exhibited a slightly higher toxicity
than BCNU alone (IC50 = 45 M) and the combination of BCNU with
O6-BG (IC50 = 30
M) (Fig. 4D). Compound 6 was also tested in
lM) was >6-fold more cytotoxic than
l
=
l
l
l
l
l
l
l
l
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G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 4. Cell survival curves for human glioma SF126, SF767 and SF763 cells treated with the indicated drugs with various concentrations. (A) SF126 cells treated with
ACNU, ACNU combined with 20
M O6-BG, and 6; (B) SF126 cells treated with BCNU, BCNU combined with 20 M O6-BG, and 6; (C) SF767 cells treated with ACNU, ACNU
combined with 20
M O6-BG, and 6; (D) SF767 cells treated with BCNU, BCNU combined with 20 M O6-BG, and 6; (E) SF763 cells treated with ACNU, ACNU combined with
20
M O6-BG, and 6; and (F) SF763 cells treated with BCNU, BCNU combined with 20 M O6-BG, and 6. The black open square, green square and red triangle represent the
M O6-BG, and treatments with 6, respectively.
l
l
l
l
l
l
treatments with ACNU or BCNU alone, combination treatments of ACNU or BCNU with 20
l
2.5. Molecular docking of human AGT inhibitors
O6-methylguanine (O6-MG) as the protein–ligand crystal complex
model. The DNA duplex in the DNA–AGT complex was removed
except for O6-MG, which was used as a native ligand for self-dock-
ing, and solvent molecules were deleted in the protein crystal
structure of 1T38. The root mean square deviation (RMSD) of the
ligand position between the docked pose with highest fitness score
and the pose in protein–ligand crystal complex was 0.0882 Å
(Fig. 7A). Therefore, the docking conformation produced by GOLD
The molecular structure of AGT used for docking studies was
acquired from homology modeling using Protein Data Bank (PDB)
entry 1T39 (3.3 Å resolution) and 1QNT (1.90 Å resolution) as tem-
plates. To validate the GOLD docking method employed in this
study, self-docking was performed using the human AGT with
PDB entry 1T38 (3.2 Å resolution) bound to DNA containing
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G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 6. The levels of dG–dC cross-link in CT DNA (A) or SF763 cells (B) treated
with ACNU (green circle), BCNU (blue square), and 6 (red triangle).
five residues and 8 or 4. Two new hydrogen-bonding interactions
were formed between Asn157 and the N3 position of guanine
and between Tyr158 and the isocyanate group of 8, although the
loss of a hydrogen bond formed between Val148 and the 2-amino
group of guanine was observed in the docked pose of 8 compared
with that of O6-BG. Moreover, compound 4 can also result in the
formation of a new hydrogen bond between the aminomethyl
group and Asn137, except for the four residues (Try114, Cys145,
Val148, and Ser159) also formed in the complex of AGT with
O6-BG. Thus, the additional interactions in the active site pocket
may increase the affinity of 8 or 4 binding to AGT and contribute
to the enhanced AGT inhibitory activity when compared with
O6-BG. Pauly et al. has demonstrated that compound 4 exhibited
better AGT inhibitory activity than O6-BG.30 This result provides
a plausible explanation for the superior cytotoxicity of 6 compared
with the combination of ACNU or BCNU with O6-BG in cells with
high AGT expression and for the high levels of dG–dC cross-link
in cells treated with 6.
Figure 5. Typical SRM ion chromatograms of dG–dC and 15N3-dG–dC internal
standard in the DNA enzymatic hydrolysates from (A) control CT DNA sample; (B)
ACNU-treated CT DNA; (C) BCNU-treated CT DNA; (D) compound 6-treated CT DNA;
(E) control cell sample; (F) ACNU-treated cells; (G) BCNU-treated cells; and (H)
compound 6-treated cells.
3. Conclusions
In this study, a new combi-nitrosourea 6, which is designed to
simultaneously release a DNA cross-linking agent and an inhibitor
of AGT, was synthesized and evaluated for antitumor activity. The
survival assay indicated that 6 exhibited more potency against
mer+ tumor cells than the clinically widely used ACNU and BCNU.
Especially, 6 was more effective in inhibiting mer+ tumor cells
growth than the combination of ACNU or BCNU with O6-BG. On
the basis of the hypothesis that the dG–dC cross-links may be used
as an indicator for predicting the anticancer efficiency of CENUs,
was similar to the crystal structure. This suggests that GOLD is
suitable for performing the docking of AGT with the corresponding
inhibitors.
Fig. 7B shows the key hydrogen bonding interaction of O6-BG
with the active pocket of AGT. Four hydrogen bonds were formed
between O6-BG and Try114, Cys145, Val148 and Ser159, which is
supported by the results of previous studies.41 The interaction of
8 and 4 with the active pocket of AGT is shown in Fig. 7C and D,
respectively. Five specific hydrogen bonds were observed between
we quantified the dG–dC cross-links induced by
6 using
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G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Figure 7. (A) Overlap of the ligand between the docked pose with the highest fitness score and the pose in the protein–ligand crystal complex; The key hydrogen bonding
interactions between O6-BG (B) or 8 (C) or 4 (D) and the amino acid residues in the active pocket of AGT. The residues forming hydrogen bonds are presented in the line model
with green representing carbon atoms. O6-BG, 8 and 4 are present in the stick model with yellow representing carbon atoms. The remaining protein is displayed in the
cartoon model.
HPLC–ESI-MS/MS to explore the relationship between the levels of
DNA cross-link and the cytotoxicity of 6. Higher levels of dG–dC
cross-link were observed in human glioma SF763 cells treated with
6 compared with cells treated with ACNU or BCNU, while the dG–
dC cross-links were observed at lower levels in CT DNA treated
with 6 than those treated with ACNU or BCNU. These combined
results suggest that the superiority of 6 may result from the AGT
inhibitory moiety, which specifically functions in mer+ cells. In
addition, the superior antitumor activity and DNA cross-linking
ability of 6 were explained by molecular docking studies. Although
6 has slightly better water solubility than O6-BG and exhibited
preferable activity compared with the traditional CENU
chemotherapies against mer+ cells, the non-selectivity to tumor
cells are likely to be a potential disadvantage hampering the fur-
ther investigations of 6 in animal experiments and clinical studies.
Therefore, we are working on the development of 6 analogs with
higher water solubility and targeting to cancer cells.
J&K Scientific Ltd. (Shanghai, China), Beijing Chemicals Co. (Beijing,
China), and TaKaRa Biotech (Tokyo, Japan) and were used without
further purification. The dG–dC cross-link standard and isotope-
labeled 15N3-dG–dC internal standard were prepared as previously
described.43,44 All of the melting points were measured with a
XT4A Electrothermal melting point apparatus and were reported
as uncorrected values. Argon was used for inert atmosphere oper-
ations in flame-dried glassware. Thin-layer chromatography (TLC)
was performed on silica gel F-254 TLC plates. Column chromatog-
raphy was performed using silica gel (200–300 mesh) with the sol-
vent mixtures specified in the corresponding experiment. 1H and
13C NMR spectra were recorded on a Bruker AV 400 M spectrome-
ter (400 MHz 1H frequency and 100 MHz 13C frequency). Chemical
shifts are reported as d (ppm) relative to tetramethylsilane as an
internal standard, and the coupling constants (J) are reported in
Hertz (Hz). IR spectra were recorded using KBr pellets on a
Bruker Vertex 70FT-IR spectrophotometer. ESI-MS spectra were
performed on a Thermo TSQ Quantum Discovery MAX triple
quadrupole mass spectrometer (Thermo Finnigan, San Jose, CA).
High-resolution mass spectra (HRMS) were obtained using a
Bruker maXis UHR-TOF mass spectrometer (Bruker Daltonics,
Bremen, Germany). HPLC-UV analysis was conducted on a Thermo
Finnigan HPLC system (Thermo Finnigan, San Jose, CA) interfaced
with a diode array detector and an autosampler.
4. Experimental section
4.1. Chemistry
All of the chemicals, solvents, reagents, and enzymes were pur-
chased from Sigma–Aldrich Chemical Co. (Milwaukee, WI, USA),
Please cite this article in press as: Sun, G.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.041

8
G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
The syntheses of compounds 2–4 and 7 were performed as pre-
(KBr): 3467, 1635, 1508, 1401, 1154 cmꢀ1. MS (ESI): m/z (M+H)+
viously described with some modifications.30
367.1. HRMS (ESI): m/z calcd for C15H13F3N6O2 (M+H)+ 367.1130,
found 367.1134.
4.1.1. 1-(2-Amino-9H-purin-6-yl)-1-methylpyrrolidinium
chloride (2)
4.1.4. O6-[3-(Aminomethyl)benzyl]guanine (4)
This compound was prepared as previously described by Kep-
pler et al.45 To a solution of 2-amino-6-chloroguanine (1.0 g,
5.9 mmol) in 40 mL anhydrous DMF at room temperature was
added 1.4 mL N-methylpyrrolidine (13.2 mmol), and the reaction
mixture was stirred for 18 h, followed by the addition of 2 mL ace-
tone to complete precipitation. The precipitates were collected on
a filter and washed twice with ether, then dried in vacuo to give
0.9 g compound 2 (3.5 mmol, yield 59%) as a white solid. Mp
200–202 °C. 1H NMR (DMSO-d6) d: 2.05–2.50 (m, 4H, –CH2-CH2–),
3.65 (s, 3H, –CH3), 3.95–4.61 (m, 4H, –CH2-N+(CH3)-CH2–), 7.11
(s, 2H, –NH2), 8.35 (s, 1H, H8), 13.44 (s, 1H, H9) ppm. 13C NMR
(100 MHz, DMSO-d6) d: 159.5, 159.03, 152.1, 143.1, 116.5, 64.6,
56.5, 52.1, 21.9, 19.0. IR (KBr): 3364, 3288, 3188, 1634 cmꢀ1. MS
(ESI): m/z (MꢀCl)+ 219.1. HRMS (ESI): m/z calcd for C10H15N6Cl
(MꢀCl)+ 219.1358, found 219.1362.
Compound 3 (300 mg, 0.82 mmol) was suspended in 34 mL
methanol along with 2 mL water containing 600 mg anhydrous
potassium carbonate (4.34 mmol), and the reaction mixture was
heated to reflux for 2 h. After evaporation of the solvents under
vacuum, the crude product was purified by column chromatogra-
phy with methanol/dichloromethane/TEA (1:5:0.05) as an eluent,
and the product was obtained as
a white solid (170 mg,
0.63 mmol, yield 77%). Mp 159–160 °C. 1H NMR (DMSO-d6) d:
3.97 (s, 2H, –CH2-NH2), 5.49 (s, 2H, –O-CH2–), 6.29 (s, 2H,
–NH2), 7.45–7.55 (m, 4H, Ar), 7.83 (s, 1H, H8) ppm, neither are
the guanine H9 proton nor the benzylic amine protons observed.
13C NMR (100 MHz, DMSO-d6) d: 160.1, 159.9, 157.7, 138.9,
137.3, 135.5, 135.4, 129.2, 129.0, 127.0, 116.2, 66.8, 46.1. IR
(KBr): 3446, 1635, 1508, 1400 cmꢀ1. MS (ESI): m/z 271.1 (M
+H)+. HRMS (ESI): m/z calcd for C13H14N6O (M+H)+ 271.1307,
found 271.1304.
4.1.2. 2,2,2-Trifluoro-N-(3-(hydroxymethyl)benzyl)acetamide
(7)
4.1.5. 1-(3-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-3-(2-
chloroethyl)urea (5)
To a solution of 4-(aminomethyl)-benzyl alcohol hydrochloride
(2.2 g, 12.6 mmol) in 15 mL anhydrous methanol was added 1.7 mL
triethylamine (TEA) (12.6 mmol) and 2.0 mL trifluoroacetic acid
ethyl ester (16.8 mmol) dropwise under argon atmosphere at room
temperature. The reaction mixture was stirred for 2 h, and then the
mixture was diluted with 20 mL ethyl acetate and 20 mL water.
The organic layer was separated, and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
washed with saturated sodium chloride and dried over anhydrous
sodium sulfate. After evaporation of the solvents under vacuum,
the crude product was purified by column chromatography with
ethyl acetate/cyclohexane (1:2) as an eluent, and the product
was obtained as white crystals (2.5 g, 10.7 mmol, yield 85%). Mp
75–76 °C. 1H NMR (DMSO-d6) d: 4.38 (d, J = 6.0 Hz, 2H, –CH2NH–),
4.49 (s, 2H, –CH2-OH), 5.21 (s, 1H, -OH), 7.13–7.32 (m, 4H, Ar),
10.00 (broad s, 1H, –NH-CO–) ppm. 13C NMR (100 MHz, DMSO-
d6) d: 156.9, 143.3, 137.8, 128.7, 126.2, 125.9, 116.5, 63.2, 43.1.
IR (KBr): 3436, 3287, 1636, 1558, 1178 cmꢀ1. MS (ESI): m/z 256.1
(M+Na)+. HRMS (ESI): m/z calcd for C10H10F3NO2 (M+H)+
234.0742, found 234.0745.
To a solution of 4 (100 mg, 0.37 mmol) in 5 mL DMF was added
0.032 mL 2-chloroethyl isocyanate (0.37 mmol) in 5 mL DMF drop-
wise at 0 °C, then the reaction mixture was stirred for 2 h at room
temperature. The reaction mixture was poured into 20 mL water
and extracted twice with 20 mL ethyl acetate. The combined
organic layers were dried over anhydrous sodium sulfate and con-
centrated under vacuum to provide a crude product, which was
reprecipitated from methanol to give 115 mg of compound 5
(0.31 mmol, yield 84%) as a white solid. Mp 150–152 °C. 1H NMR
(DMSO-d6) d: 3.31–3.72 (m, 4H, –CH2-CH2-Cl), 4.22 (d, J = 6.0 Hz,
2H, –CH2-NH–), 5.50 (s, 2H, –O-CH2–), 6.28 (s, 1H, –CO-NH–),
6.62 (s, 1H, –NH-CO–), 7.23–7.43 (m, 4H, Ar), 7.94 (s, 1H, H8)
ppm, the guanine H9 proton was not observed. 13C NMR
(100 MHz, DMSO-d6) d: 159.9, 159.8, 158.4, 156.1, 154.2, 141.5,
139.3, 136.9, 128.8, 127.6, 127.5, 127.2, 67.6, 45.0, 43.3, 42.0. IR
(KBr): 3446, 1635, 1508, 1399 cmꢀ1. MS (ESI): m/z 375.9 (M+H)+
with 35Cl, 377.9 [M+H]+ with 37Cl. HRMS (ESI): m/z calcd for
C
16H18ClN7O2 (M+H)+ 376.1289, found 376.1284.
4.1.6. 3-(3-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-
chloroethyl)-1-nitrosourea (6)
4.1.3. N-(3-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-2,2,2-
trifluoroacetamide (3)
To a solution of 5 (100 mg, 0.27 mmol) in 3 mL anhydrous ace-
To a solution of 7 (1.18 g, 5.0 mmol) in 20 mL anhydrous DMF
was added 1.2 g potassium t-butoxide (10.7 mmol) followed by
600 mg 2 (2.36 mmol) under an argon atmosphere. The reaction
tonitrile containing 23.2 lL glacial acetic acid (0.405 mmol) was
added 47.3 mg nitrosonium tetrafluoroborate (0.405 mmol), and
the reaction mixture was stirred for 3 h at 0 °C until disappearance
of the starting material as monitored by TLC. The reaction mixture
was poured into 30 mL ice-cold water and extracted twice with
30 mL ice-cold ethyl acetate. The combined organic layers were
washed with saturated sodium chloride, dried over anhydrous
sodium sulfate and concentrated under vacuum below 30 °C. The
crude product was purified by column chromatography with ethyl
acetate/petroleum ether (5:1) as an eluent to give the final product
6 (66 mg, 0.16 mmol, yield 59%). Mp 135–137 °C. 1H NMR (DMSO-
d6) d: 3.31–3.59 (m, 4H, –CH2-CH2-Cl), 4.23 (d, J = 5.6 Hz, 2H,
–CH2-NH–), 5.50 (s, 2H, –O-CH2–), 6.43 (s, 2H, –NH2), 6.97–7.36
(m, 4H, Ar), 7.96 (s, 1H, H8), 9.18 (s, 1H, –NH-CO–). 13C NMR
(100 MHz, DMSO-d6) d: 153.7, 152.3, 137.1, 135.7, 129.2, 129.1,
128.1, 128.0, 127.8, 127.6, 127.4, 67.8, 43.3, 43.2, 42.7. IR (KBr):
mixture was stirred for 4 h, and then 440 lL glacial acetic acid in
14 mL water was added to neutralize the excess potassium t-
butoxide. The mixture was diluted with 30 mL ethyl acetate and
30 mL saturated ammonium chloride. The organic layer was sepa-
rated, and the aqueous layer was extracted twice with ethyl acet-
ate. The combined organic layers were dried over anhydrous
sodium sulfate and concentrated under vacuum. The crude product
was purified by column chromatography, eluted first with metha-
nol/dichloromethane (1:50) followed by methanol/dichloro-
methane (1:10), and the product was obtained as a white solid
(450 mg, 1.2 mmol, yield 51%). Mp 220–222 °C. 1H NMR (DMSO-
d6) d: 4.42 (d, J = 5.6 Hz, 2H, –CH2-NH–), 5.49 (s, 2H, –O-CH2–),
6.30 (s, 2H, –NH2), 7.27–7.44 (m, 4H, Ar), 7.80 (s, 1H, H8), 10.02
(broad s, 1H, –NH-CO–), 12.43 (broad s, 1H, H9) ppm. 13C NMR
(100 MHz, DMSO-d6) d: 160.1, 156.9, 143.3, 138.2, 137.6, 129.1,
128.7, 127.9, 127.5, 126.4, 126.2, 125.9, 116.5, 67.1, 43.0. IR
3447, 1635, 1507, 1400 cmꢀ1
with 35Cl, 406.9 (M+H)+ with 37Cl. HRMS (ESI): m/z calcd for
16H17ClN8O3 (M+H)+ 405.1190, found 405.1194.
.
MS (ESI): m/z 404.9 [M+H]+
C
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G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
4.2. Stability
4.5. In vitro treatment of calf thymus DNA with 6
The degradation of 6 was monitored using a Thermo Finnigan
HPLC system (Thermo Finnigan, San Jose, CA) interfaced with a
diode array detector and an autosampler. The analytes were sepa-
Calf thymus DNA (CT DNA) was dissolved in phosphate buffered
saline (10 mM Tris, 50 mM NaCl, 50 mM Na2HPO4, pH 7.4) with a
final concentration of 1 mg/mL. A freshly prepared solution of 6
(dissolved in DMSO) was added to the CT DNA solution to give a
final concentration of 0.1, 0.2, and 0.4 mM. The reaction mixture
was incubated at 37 °C with a circulating water bath for 12 h. Ali-
rated using a Phenomenex Luna C18 reverse phase column (5 lm,
250 ꢁ 4.6 mm, Phenomenex, Torrance, CA) eluted with 50% 50 mM
ammonium acetate (pH 4.5, solvent A) and 50% methanol (solvent
B) at a flow rate of 1.0 mL/min. Compound 6 was dissolved in a
minimum volume of dimethyl sulfoxide (DMSO) and diluted with
PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 dis-
solved in 1000 mL deionized water, pH 7.4) or MEM-EBSS supple-
mented with 10% fetal bovine serum (FBS) and antibiotics (100 U/
quots of 400
tion. For each sample, the DNA was precipitated by the addition
of 800 ice-cold ethanol followed by centrifugation at
lL of reaction solution were removed for DNA isola-
lL
12,000 rpm for 5 min at 4 °C. The resulting DNA pellets were
washed with 1 mL of 75% ethanol and then with 1 mL of 100% etha-
nol. All DNA samples were dried under a nitrogen stream and
stored at ꢀ20 °C until enzymatic hydrolysis. The CT DNA samples
treated with ACNU (dissolved in deionized water) and BCNU (dis-
solved in ethanol) were regarded as positive controls. The negative
control DNA samples were incubated under the same conditions as
the treated DNA samples, except without the addition of drugs.
mL penicillin and 100
tration of 0.1 mM. The mixtures were incubated at 37 °C with a cir-
culating water bath, and 200 L of solution was removed at the
lg/mL streptomycin) to give a final concen-
l
indicated time point and stored at ꢀ20 °C until HPLC analysis. In
PBS, all of the samples were injected for HPLC-UV analysis without
further treatments. In MEM-EBSS, the cellular and medium compo-
nents in the samples were precipitated by adding an equal volume
of acetonitrile followed by centrifugation at 12,000 rpm for 5 min.
The supernatants were removed for HPLC-UV analysis.
4.6. Treatment of human glioma SF763 cells with 6
The human glioma SF763 cells were plated at a density of 106
cells in T75 culture flasks and were allowed to grow to reach
ꢂ90% confluence. Then, the medium was discarded and replaced
with freshly prepared medium containing different concentrations
of 6 (0.1, 0.2, and 0.4 mM). After incubation for 12 h, the medium
was discarded, and the cells were washed twice with D-Hanks’
basal salt solution. The cells were digested with 0.25% trypsin con-
taining 0.02% versene and collected by centrifugation at 1000 rpm
for 10 min. All cell samples were stored at ꢀ20 °C until DNA isola-
tion. The cells treated with ACNU and BCNU were regarded as pos-
itive controls. The negative control cell samples were incubated
under the same conditions as the treated cell samples, except
without the addition of drugs.
4.3. Cell culture
The human glioma SF126 (merꢀ, low or no AGT activity), SF767
(mer+, 61 fmol/106 cells) and SF763 (mer+, 119 fmol/106 cells)39,40
cells were obtained from Peking Union Medical College and were
maintained in MEM-EBSS supplemented with 10% FBS and antibi-
otics (100 U/mL penicillin and 100 lg/mL streptomycin) at 37 °C in
a humidified atmosphere of 5% CO2/95% air. The cells were
passaged as required every 2–3 days.
4.4. Cell survival assay
4.7. DNA isolation
The cell survival assay was performed using the Cell Counting
Kit-8 (CCK8, Dojindo China CO., Ltd, Shanghai, China) in 96-well
plates. Cells were plated at a density of 5000 cells/well in 96-well
plates and allowed to grow for 24 h. Then, the medium was dis-
carded and replaced with fresh medium containing different con-
centrations of drugs for a 24 h incubation. For the combination
treatments, O6-BG was added to give a final concentration of
DNA was isolated from cells using the procedure previously
described with some modifications.22,46,47 Briefly, the cell samples
were suspended in 10 mL cell lysis buffer (10 mM Tris–HCl, 0.1 M
EDTA, and 0.5% SDS, pH 8.0), then 70
lL protease K (21 U) and
100 L RNase A (0.5 mg) were added. The mixture was incubated
l
at 37 °C overnight with shaking followed by a standard phenol–
chloroform extraction procedure. The supernatant containing
DNA was collected, and an equal volume of ice-cold isopropanol
was added to precipitate DNA. After centrifugation at 12,000 rpm
for 10 min, the DNA pellets were washed with 1 mL of 75% ethanol
and then with 1 mL of 100% ethanol. All DNA samples were dried
under a nitrogen stream and stored at ꢀ20 °C until enzymatic
hydrolysis.
20 lM and was incubated for 2 h before exposure to ACNU or
BCNU. After 2 h, the medium was replaced with fresh medium con-
taining different concentrations of ACNU or BCNU for an additional
24 h. Then, the medium containing different concentrations of
drugs was discarded, and 100 lL fresh medium containing 10%
CCK8 was added to each well. After incubation for 3 h, the absor-
bance was determined using a Thermo Scientific Multiskan FC
(Waltham, MA) at 450 nm. The cell survival rate (%) was expressed
by the following formula:
4.8. DNA enzymatic hydrolysis
The DNA enzymatic hydrolysis was performed as previously
described with some modifications.22,44 DNA samples were redis-
solved in Tris–HCl buffer (10 mM, pH 7.4). The purity of the iso-
lated DNA samples from SF763 cells was determined by the
absorbance ratio A260/A280, which was typically between 1.7
and 2.0. Four enzymes, including recombinant deoxyribonuclease
I (DNaseI), S1 nuclease, alkaline phosphatase (CIAP, from calf intes-
tine), and phosphodiesterase I (from Crotalus adamanteus venom),
were used to digest DNA into single nucleotides. Briefly, a solution
Survival rateð%Þ ¼ ðAdrug ꢀ AblankÞ=ðAcontrol ꢀ AblankÞ ꢁ 100%:
where Adrug represents the absorbance of the wells containing the
medium, cells and drug, Ablank represents the absorbance of the
wells containing only the medium, and Acontrol represents the absor-
bance of the wells containing the medium and cells. The results
were expressed as the mean values standard deviation (SD) from
at least three independent experiments. The final concentration of
ethanol (BCNU was dissolved in ethanol) or DMSO (6 and O6-BG
was dissolved in DMSO) in culture medium was below 0.1%.
of 100
the mixture was heated at 98 °C for 5 min followed quickly by chil-
ling in an ice-bath for 10 min. Then, 15 L DNase I (3 U/ L) and
l
L DNA was spiked with 15N3-dG–dC internal standard, and
l
l
Please cite this article in press as: Sun, G.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.041

10
G. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
10
l
L S1 nuclease (10 U/
L CIAP (1 U/
L) were further added to the mixture for incubation at
l
L) were added for incubation at 37 °C for
5.2 software (Cambridge Structural Database System) was selected
6 h. Subsequently, 20
I (0.001 U/
37 °C overnight. Finally, enzymes and undigested DNA were
removed by a Microcon YM-10 molecular weight centrifugal filter
(Billerica, MA). The final concentration of the 15N3-dG–dC internal
standard in the filtrate was 9.6 nM. A 10 lL aliquot was removed
for dGuo quantitation, and approximately 140
remained for HPLC–ESI-MS/MS analysis.
l
l
L) and 5 L phosphodiesterase
l
for the docking simulations. Self-docking was performed to validate
the docking method using the X-ray crystal structure of human AGT
with PDB entry 1T38 (3.2 Å resolution) bound to DNA containing
O6-MG as the protein–ligand crystal complex model. For self-dock-
ing, DNA was removed, except for O6-MG, which was used as the
native ligand for self-docking. Solvent molecules were removed in
the X-ray crystal structure of 1T38. Geometry optimizations of
the ligands were performed at the B3LYP/6-31g+(d,p) theoretical
level using Gaussian 09 software (Gaussian Inc., Wallingford, CT,
USA).50 Hydrogen atoms were added to the modeled protein, and
the Gold score was selected as the scoring function.
l
lL of filtrate
4.9. dGuo quantitation
The DNA concentration was quantified by HPLC-UV analysis for
dGuo in the enzymatic hydrolysates. The quantitation of dGuo was
conducted using a Thermo Finnigan HPLC system (Thermo Finni-
gan, San Jose, CA) incorporating a diode array detector and an
autosampler. A Phenomenex Luna C18 reverse phase column
Acknowledgments
The authors would like to thank Dr. Stephen S. Hecht in the
University of Minnesota for his valuable suggestions to the study.
This study was supported by grants from the National Natural
Science Foundation of China (No. 21277001), Natural Science
Foundation of Beijing (No. 7162015), Beijing Municipal Education
(5
lm, 250 ꢁ 4.6 mm, Phenomenex, Torrance, CA) was used for
dGuo separation with deionized water (solvent A) and methanol
(solvent B) as the mobile phases at a flow rate of 0.7 mL/min.
The gradient elution was performed as previously described.22
The UV wavelength was monitored at 254 nm. The quantitation
was based on the HPLC peak area of dGuo using an external cali-
bration curve constructed by plotting the HPLC peak area of the
dGuo standard versus the corresponding concentration.
Commission
Science
and
Technology
Project
(No.
PXM2015_014204_500175), and the Jinghua Talent Project of
Beijing University of Technology (015000514115001).
A. Supplementary data
4.10. HPLC–ESI-MS/MS analysis of dG–dC cross-links
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.03.041.
The quantitation of dG–dC cross-links in DNA enzymatic hydro-
lysates was performed using a Thermo TSQ Quantum Discovery
MAX triple quadrupole mass spectrometer (Thermo Finnigan, San
Jose, CA) interfaced with an HPLC system. A Zorbax SB C18 reverse
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phase column (2.1 ꢁ 150 mm, 5
lm particle size; Agilent Tech-
nologies, Palo Alto, CA) was eluted with deionized water contain-
ing 0.01% acetic acid (solvent A) and acetonitrile (solvent B) at a
flow rate of 0.2 mL/min. The solvent composition was maintained
with 5% B at 0 min and then linearly increased to 10% B in
23 min. Subsequently, solvent B was decreased to 5% over 2 min
followed by
a 10 min equilibration. Enzymatic hydrolysate
(15 L) was injected for HPLC–ESI-MS/MS analysis. The mass spec-
l
trometer parameters were optimized using the 15N3-dG–dC inter-
nal standard. The ESI source was operated in the positive ion
mode with argon as the collision gas (1.5 mTorr). Nitrogen was
used as the sheath gas (50 psi) and the auxiliary gas (5 psi). The
spray voltage was 3.5 kV, and the capillary temperature was
300 °C. The tube lens offset, source collision induced dissociation
(CID) and collision energy were set at 121 V, 10 V and 20 eV,
respectively. The SRM mode was used for quantitative analysis of
dG–dC cross-links by monitoring the transitions of m/z 521 ?
289 for dG–dC and m/z 524 ? 292 for the 15N3-dG–dC internal
standard. The levels of dG–dC cross-link were expressed as fmol
of dG–dC per mg DNA. The amount of DNA was calculated from
the dGuo content by considering that 1 mg of DNA contains 3 lmol
of nucleotides and that dGuo accounts for 22% of the total nucleo-
tides in DNA.48,49 The results were expressed as the mean
values standard deviation (SD) from triplicate experiments.
4.11. Molecular docking study
Two X-ray crystal structures of the AGT protein with PDB entry
1T39 and 1QNT were used as templates to model a new AGT crystal
structure (NCBI Reference Sequence: NP_002403.2) using the
MODELER section in Discovery Studio 4.0 (Accelrys Inc.). The con-
formational rationality of the modeled AGT protein was evaluated
by the Ramanchandran Plot and the Line Plot shown in Figures S3
and S4, respectively, in the Supplementary materials. GOLD Suite
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Please cite this article in press as: Sun, G.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.041