A thiol-inducible and quick-response DNA cross-linking agent

Article abstract of DOI:10.1016/j.bmcl.2018.11.040

Three new 2,4-dinitrobenzenesulfonyl derivatives 1–3 were successfully prepared for the first time using a simple process. They were efficiently triggered by thiols (glutathione and L-cysteine) to release the corresponding phenol derivatives (4–6) within

Full text of DOI:10.1016/j.bmcl.2018.11.040

Accepted Manuscript
A thiol-inducible and quick-response DNA cross-linking agent
Yuanzhen Xu, Hongbo Wei, Jianjun Chen, Kun Gao
PII:
S0960-894X(18)30907-7
https://doi.org/10.1016/j.bmcl.2018.11.040
BMCL 26151
DOI:
Reference:
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 September 2018
14 November 2018
18 November 2018
Please cite this article as: Xu, Y., Wei, H., Chen, J., Gao, K., A thiol-inducible and quick-response DNA cross-
linking agent, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.040
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
A thiol-inducible and quick-response DNA cross-linking agent
a,b
b
a
a
Yuanzhen Xu, Hongbo Wei, Jianjun Chen and Kun Gao*
a
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Gansu, Lanzhou, 730000, China.
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Shaanxi, Yangling, 712100,
b
China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Three new 2,4-dinitrobenzenesulfonyl derivatives 1–3 were successfully prepared for the first
time using a simple process. They were efficiently triggered by thiols (glutathione and L-
cysteine) to release the corresponding phenol derivatives (4–6) within 5 minutes. The quick
1
response of 1–3 toward thiols was determined by H NMR and HPLC. Moreover, our results
Available online
indicated that 1 could induce DNA cross-linking in the presence of glutathione, probably due to
the quinone methide formation of phenol intermediate 4 followed by departure of 2,4-
dinitrobenzenesulfonyl group.
Keywords:
quinone methide
DNA cross-linking
antitumor agents
2
, 4-dinitrobenzenesulfonyl
2
009 Elsevier Ltd. All rights reserved.
Antitumor agents that target DNA, such as nitrogen mustard,
mitomycin C, and psoralen, prevent the melting of DNA double
1
strands, disrupt cell maintenance and transcription, and finally
lead to cell death. However, the side effects due to nonspecific
targeting limit the clinical application of these agents. Therefore,
induced DNA cross-linking agents are playing increasingly
2
important roles in cancer therapy. Among these agents, quinone
methides (QMs) are important intermediates for the ultimate
3
cytotoxins responsible for the antitumor activity. Moreover, they
can be generated by many methods, including photochemical,
fluoride-induced activation, thermal digestion, oxidation, and
4
Figure 1. The structure of compounds 1–6.
H
2
O
2
-induced reactions. Unfortunately, most of these methods
increase the occurrence of side effects due to undesirably high
reaction temperatures, the requirement for additional reagents
and acidic or basic conditions, long reaction times, and the
New compounds 1–3 and three known compounds 4-6 were
synthesized starting from the corresponding commercially
available phenol derivatives according to the procedure reported
previous. For example, compound 7 was obtained by Mannich
reaction with hydroquinone as the starting material (Scheme 1).
5
inaccessibility of precursors. One efficient strategy to reduce the
10
toxicity of inducible quinone methide precursors would be the
production of quick-response reactions toward the bioavailable
molecule under tumor-specific conditions. Glutathione (GSH) is
Treatment of 7 with 2,4-dinitrobenzene sulfonyl chloride yielded
8
, quaternarization of which was carried out by reaction with
6
the most abundant free thiol in the cell and has been shown to be
methyl iodide to prepare compound 1. Quaternarization of 7
yielded compound 4.
First, the inducible reactivity was investigated. To examine all
intermediates formed, treatment of 1–3 with L-cysteine (L-Cys),
more abundant in tumors than in the corresponding normal
7
tissue. Moreover, GSH has been reported to efficiently activate
2
,4-dinitrobenzenesulfonyl derivatives.8 Our group recently
demonstrated that an anticancer prodrug coupled with a 2,4-
dinitrobenzenesulfonyl group could be triggered by GSH and
release the chemotherapeutic agent mechlorethamine.9
Considering that QM intermediate could be induced by a phenol
which is the active group of GSH, was carried out in a mixture of
1
D
2
O and DMSO-d
6
by H NMR analysis. However, the reaction
was so fast that compounds 1–3 were completely consumed
within 5 minutes (Fig. S1 of the Supplementary file) and
converted to the final product, corresponding to the phenol
derivatives 4–6 (Fig. 1). Moreover, MS analysis of the reaction
mixture of compounds 1–3 with L-Cys also showed the release of
the corresponding phenol derivatives (Fig. S2-S4 of the
Supplementary file). According to the previously reported
mechanism of 2, 4-dinitrobenzenesulfonyl group as leaving
4
f
quaternary ammonium structure , we design and investigate new
inducible reactivities of the 2,4-dinitrobenzenesulfonyl
compounds 1–3 (Fig. 1).

Corresponding author. E-mail: npchem@lzu.edu.cn

8
d
group upon reaction with glutathione (GSH) , the reaction was
triggered by L-Cys and produced the intermediates 1a–3a, which
were immediately converted to the phenol derivatives 4–6
through elimination of SO
Supplementary file).
selective for cellular thiols (GSH and L-Cys) over other amino
acids.
The DNA cross-linking abilities of compounds 1–3 were next
investigated in phosphate buffer (pH 7.3) using linearized
plasmid DNA (pBR322) by denaturing alkaline agarose gel
2
(Scheme 2, Scheme S1 of the
4
a,4b,11
electrophoresis (Fig. 3).
Our previous study indicated that
9
GSH produced no DNA cross-linking. However, treatment of
DNA with compound 1 in the presence of GSH induced about 34%
DNA cross-linking at 1 mM, similar to that induced by
compound 4 (43%) under the same conditions. These results
indicated that the designed compound 1 could be considered as a
thiol-triggered DNA cross-linking agent through a quinone
methide intermediate generated by compound 4. Rokita and co-
1
2
workers showed that the adducts of electron-rich o-QMs with
6
2
dA N and dG N1, N remained stable over the course of
observation. Moreover, the N7 adduct of dG was easily
deglycosylated to form its guanine derivative, which was also
stable. Therefore, the DNA cross-linking sites induced by 4
through o-QM 4a and 4b (Fig. 4) with an electron-donating
hydroxyl group most likely occurred at dA and dG. Meanwhile,
compounds 2 and 3 showed no DNA cross-linking properties.
Peng and colleagues reported that the stable phenol product 6
Scheme 1. Synthesis of compounds 1 and 4: a) 33% aqueous (CH
formaldehyde aqueous solution, 30°C for 30 min, then 90-95°C 2 h; b) 2,4-
dinitrobenzene sulfonyl chloride, K CO , THF, RT; c) CH I, CH CN, RT.
3 2
) NH, 37%
2
3
3
3
4
f
produced from 3 did not undergo QM formation. However, the
product 5 obtained from 2, which shared a five-atom bridge
1
3,14
between alkylation sites common to N-mustards,
to be capable of inducing DNA cross-linking.
was shown
4
b,15
To determine
whether QM was generated from compound 2 by triggering with
L-Cys, a QM trapping experiment with a large excess of ethyl
vinyl ether (EVE) was performed (Scheme 3). Compound 13
(m/z=264.1960) was detected by HPLC and HR-ESIMS when 2
was incubated at 37°C for 24 hours in the presence of L-Cys and
EVE, suggesting the formation of QM. The lack of DNA cross-
link formation by compound 2 was probably due to two factors.
First, due to the unstablity of 2 in phosphate buffer compared to 1
and 3 (Fig. S5-S7 of the Supplementary file), the QM generated
from 2 could initially react with a range of cellular components,
Scheme 2. The mechanism of the reaction of compound 1 and L-Cys.
Although sulfonyl esters were hydrolyzed to some extent
under physiological conditions (Fig. S5-S7 of the Supplementary
1
file), H NMR analysis suggested that compounds 1–3 could also
release the corresponding phenol derivatives 4–6 within 5
minutes in the presence of L-Cys or GSH (Fig. S8 of the
Supplementary file). HPLC and MS analysis were performed to
further evaluate the quick response of 1–3 toward thiols. Of
several substances tested, coumarin was chosen as the most
appropriate internal standard (IS) in this assay because it was
stable and did not interfere with the product of the inducible
2
such as H O and L-Cys, to form kinetic products (Scheme S2 of
the Supplementary file). Second, electronic perturbation of QM
markedly influences the stability and, in turn, alters the kinetics
and product profile of the QM reaction with deoxynucleosides. A
previous study showed that a related adduct with an electron-
12
donating methyl group is very labile and regenerates its QM.
reactions. The results indicated that the thioether (2-amino-3-[S-
+
(
2,4-dinitrobenzene)]propionic acid, [M+H ]=288, t≈17 min) was
generated within 2 minutes after addition of compounds 1–3 (Fig.
S9 of the Supplementary file). Moreover, the percentage of
thioether compared with that of coumarin did not change
markedly over a period of 4.5 hours (Table S1 of the
Supplementary file). The reaction mixture without L-Cys or
compounds 1–3 had a light yellow or achromatic color.
Interestingly, the phosphate buffer of L-Cys immediately showed
a brilliant red color after the addition of compounds 1–3, and it
showed an orange color 1 minute later (Fig. 2, Fig. S10 of the
Supplementary file), indicating the reactions of compounds 1–3
with L-Cys. Taken together, these results indicated that the
inducible reaction of compounds 1–3 by L-Cys was
instantaneous, and these three compounds could be used as
quick-response agents toward thiols.
Figure 2. Analysis of color change during the reaction of compound 1 with
L-Cys in phosphate buffer (pH 7.3): (1) 5 mM 1; (2) 5 mM 1 + 10 mM L-Cys;
(3) 2.5 mM 1; (4) 2.5 mM 1 + 5 mM L-Cys; (5) 1 mM 1; (6) 1 mM 1 + 2 mM
L-Cys.
To obtain further insight into the selectivity of the reaction,
we studied the inducible activity of 1 toward other amino acids,
such as L-arginine, L-proline, β-alanine, glycine, and L-valine
(Fig. S11 of the Supplementary file). The results indicated that
the production of 4 was not triggered by other amino acids,
suggesting that the activation of 1 to release 4 was highly
Figure 3. DNA cross-linking ability of compounds 1-6 in phosphate buffer
(pH 7.3). Lane 1, pBR322 (control); lane 2, pBR322 + 2 mM GSH + 1 mM 1

(
cross-linking yield 34%); lane 3, pBR322 + 1 mM 4 (43%); lane 4, pBR322
1 mM GSH + 1 mM 2 (0%); lane 5, pBR322 + 1 mM 5 (0%); lane 6,
pBR322 + 2 mM GSH + 1 mM 3 (0%); lane 7, pBR322 + 1 mM 6 (0%).
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Scheme 3. Trapping reaction in the presence of EVE.
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8
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Figure 4. Tandem QM generation and DNA cross-linking formation induced
by 1 upon RSH activation.
1
1
1
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In conclusion, we synthesized three thiol-inducible 2,4-
dinitrobenzenesulfonyl derivatives. Among them, compound 1
could be efficiently triggered by thiols and released the
corresponding phenol derivative 4, which directly produced QM
and induced DNA cross-linking under physiological conditions.
Its quick response to thiols makes compound 1 the preferred lead
compound for developing novel quinone methide precursors as
selective DNA cross-linking agents. To further increase the
selectivity and stability of compound 1, we will attempt to
introduce a cancer targeting unit such as biotin and other benzylic
leaving groups, such as dimethyl amine, in the trimethyl amine
position in future studies.
2
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1
Supplementary Material
Acknowledgments
This work was supported by the National Natural
Science Foundation of China (No. 21002046 and 31270396).
Conflicts of interest
There are no conflicts to declare.
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1
2
3
4
. Three new 2, 4-dinitrobenzenesulfonyl derivatives 1–3 were successfully prepared for the first time.
. The inducible reactions of 1–3 were immediately triggered by RSH.
. The inducible activity of 1 was highly selective for RSH over other amino acids.
. Compound 1 could release of the corresponding phenol derivative and induce DNA cross-linking.
S2