Biological studies of photoinducible phenol quaternary ammonium derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.12.007

Three water-soluble DNA cross-linking phenol quaternary ammonium derivatives 3, 4, and 5 could inhibit the transcription in vitro by photoactivation. DNA interstrand cross-linking action might be the key factor to inhibit transcription by these compounds.

Full text of DOI:10.1016/j.bmcl.2005.12.007

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1660–1664
Biological studies of photoinducible phenol quaternary
ammonium derivatives
b,
Yang Song,a, Ping Wang,a, Juanjuan Wu,b Xiang Zhou,a,c,e, Xiao-Lian Zhang,
*
*
Linhong Weng,d Xiaoping Caoe and Feng Lianga
aCollege of Chemistry and Molecular Sciences, Wuhan University, Hubei, Wuhan 430072, PR China
bSchool of Medicine, Wuhan University, Hubei, Wuhan 430071, PR China
cKey Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education,
Peking University, Beijing 100871, PR China
dDepartment of Chemistry, Fudan University, Shanghai 200433, PR China
eState Key Laboratory of Applied Organic Chemistry, Lanzhou University, Gansu, Lanzhou 730000, PR China
Received 15 September 2005; revised 27 November 2005; accepted 6 December 2005
Available online 27 December 2005
Abstract—Three water-soluble DNA cross-linking phenol quaternary ammonium derivatives 3, 4, and 5 could inhibit the transcrip-
tion in vitro by photoactivation. DNA interstrand cross-linking action might be the key factor to inhibit transcription by these com-
pounds. Further tumor cell apoptosis was observed by flow cytometry it indicated that cross-linking agent 5 could significantly
induce the late apoptosis of tumor cells.
Ó 2005 Elsevier Ltd. All rights reserved.
Transcription is a critical step in protein synthesis. Dur-
ing this cellular process, mRNA is assembled from a
DNA template.1 As the inhibition of transcription could
induce coding of RNA and proteins incompletely and
then lead to cell death finally, it has been found to be
potentially useful in the treatment of various cancers
and searching for antiviral or anti-tumor agents such
as cisplatin, actinomycin, etc.2–5 The active functions
of some such agents are involved in DNA interstrand
cross-linking mechanism.6–8 It has been speculated that
DNA interstrand cross-links might affect cells by dis-
rupting transcription.9 Meanwhile, photocrosslinks of
DNA by irradiation under mild conditions and without
any additives such as metals, oxidative agents, and
reducing agents will offer considerable potential applica-
tion in medicine.6 Recently, we have reported a biphenol
diquaternary ammonium 5 that is a potent, water-solu-
ble, and photoinducible DNA cross-linking agent.10
Electrophoresis of the reaction mixtures showed that
compound 5 was 100-fold more efficient than compound
4 at effecting DNA cross-linking. Cross-linked DNA
was present at concentrations of compound 5 as low
as 1.0 lM.10 Under irradiation, it could form an o-qui-
none methide (o-QM) intermediate, which is an active
electrophile in biological and chemical processes,11–13
and then induce DNA cross-link. More recently, Frecce-
ro’s group found that binol quaternary ammonium
derivatives could cross-link DNA under illumination
by the benzo-QM pathway.14 In the present work, we
report our new findings about their inhibitions of
transcription in vitro by phenol quaternary ammonium
derivatives 3, 4, and 5. We have found that DNA
interstrand cross-linking action might be the key factor
to inhibit transcription. Furthermore, we have observed
that compound 5 could induce tumor cell apoptosis,
which could be potentially available modulating drugs
for treating cancer in future.
To investigate the structure–activity relationship of
DNA cross-linking abilities and inhibition of transcrip-
tion, we chose compounds 3, 4, and 5 as our studied sub-
strates. Synthesis of compounds 4 and 5 was reported in
our previous paper.10 1,4-Dihydroxyl-benzene diquater-
nary ammonium 3 was a biphenol diquaternary ammo-
nium analogue and had a similar structure as compound
Keywords: DNA cross-linking agent; Photoinducible; Interstrand;
Apoptosis.
*
Corresponding authors. Tel.: +86 27 61056559; fax: +86 27
87336380; e-mail addresses: zhouxiang65@hotmail.com; zhangxl65@
hotmail.com
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.007

Y. Song et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1660–1664
1661
A
1
2
3
4
mRNA
B
C
1
2
3
4
mRNA
1
2
3
4
mRNA
Figure 1. (A) Ethidium bromide-stained agarose gel (0.9%) of
transcribed mRNA by compound 5. Lane 1, from DNA template
(0.05 lg); lane 2, from DNA template (0.05 lg) + hm (30 min); lane 3,
from DNA template (0.05 lg) + 5 (5 lM); lane 4, from DNA template
(0.05 lg) + 5 (1 lM) + hm (30 min). (B) Concentration dependence of 5
for transcribed mRNA (ethidium bromide-stained agarose gel (0.9%)).
Lane 1, from DNA template (0.05 lg) + 5 (5 lM); lane 2, from DNA
template (0.05 lg) + 5 (0.2 lM) + hm (30 min); lane 3, from DNA
template (0.05 lg) + 5 (1 lM) + hm (30 min); lane 4, from DNA
template (0.05 lg) + 5 (5 lM) + hm (30 min). (C) Comparison of
compounds 3, 4, and 5 for transcribed mRNA (Ethidium bromide-
stained agarose gel (0.9%)). Lane 1, from DNA template (0.05 lg);
lane 2, from DNA template (0.05 lg) + 4 (10 lM) + hm (30 min); lane
3, from DNA template (0.05 lg) + 3 (10 lM) + hm (30 min); lane 4,
from DNA template (0.05 lg) + 5 (10 lM) + hm.
Scheme 1. Synthesis of 2 and 3.
5. Compound 3 was prepared by methylation of com-
pound 216 in CH3CN with the yield of 71% (Scheme
1). It was fully characterized by H NMR, 13C NMR,
and elemental analysis.
1
Inhibition of transcription by compounds 3, 4, and 5
was tested. The experiments selected target DNA con-
sisting of T7 promoter that was in a completely con-
trolled in vitro transcription system by T7 RNA
polymerase.15 Prior to transcription, the target DNA
was mixed to a given concentration of DNA cross-link-
ing agents 3, 4 and 5, respectively, at room temperature
and was irradiated for 30 min using a 50-W high-pres-
sure mercury lamp at the distance of 20 cm. Transcrip-
tion with T7 RNA polymerase was performed
according to the protocol recommended by Promega
(P1300). For the control experiment, RNA was synthe-
sized in the dark or under the irradiation of target
DNA alone in the absence of drug or in the presence
of drug without illumination. The results are shown in
Figure 1.
The twisted form and two quaternary ammonium
groups could anchor DNA strands and facilitate bind-
ing of DNA strands that cross-linking between strands
then proceeded through a bis(quinone methide) interme-
diate.16 However, for compounds 3 and 4, they might
involve in tandem formation of o-quinone methide
intermediate13 under illumination after they bound to
DNA. Therefore, compound 5 might cross-link DNA
with ease after photoactivation.
Our further demonstration about existence of bis(qui-
none methide) intermediate was performed by trapping
reaction with tert-butyl amine under illumination
(Scheme 2). Stronger nucleophilic agent tert-butyl amine
could react with bis(quinone methide) intermediate and
form secondary amine 6. Further DNA cross-linking
experimental data indicated those of, compared with
compounds 3 and 4, compound 5 had the strongest abil-
ity to cross-link DNA and compound 4 had the weakest
ability (gel not shown here).
Concentration dependence of 5 for inhibition of tran-
scription had also been investigated (Fig. 1B). The inhi-
bition of transcription increased with the concentration
of compound 5 relative to the target DNA. At a concen-
tration of 5 lM, the compound 5 could almost inhibit
the transcription completely (Fig. 1B, lane 4). We had
compared their abilities to inhibit transcription by com-
pounds 3, 4, and 5 and the results are shown in Figure
3C. At a concentration of 10 lM, compound 4 had no
significant inhibition (Fig. 1C, lane 2), and compound
3 had significant ability of inhibition (Fig. 1C, lane 3).
However, at this concentration, compound 5 completely
inhibited the transcription process at all (Fig. 1C, lane
4). These results indicated that compound 5 had appar-
ently the best ability to inhibit transcription among
three agents.
DNA cross-linked by these compounds might be one
important factor to inhibit transcription. As the most
efficient DNA cross-link agent, compound 5 could
strongly prohibit the formation of the ‘open’ structure
of DNA by holding two strands together. Therefore, it
might possess the strongest inhibition ability of tran-
scription among them. The transcription abilities of

1662
Y. Song et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1660–1664
Scheme 2. Trapping reaction of bis(quinone methide) intermediate.
inhibition corresponded also to their DNA cross-linking
formations.6 Apparently, interstrand DNA cross-link is
the key step to block DNA strand opening and then
inhibition of transcription occured.6
Scheme 3. Proposed mechanism of DNA cross-linking agents.
(bases), 4363 + 2686 (bases) and 4363 + 4363 (bases).
Our experimental data clearly indicated that only two
To identify DNA–DNA cross-link by compound 5 in-
volved in interstrand formation, we mixed both linearized
pBR322 (4363 bp) and pUC19 (2686 bp), and checked
them by denaturing alkaline agarose gel electrophoresis
reported by Cech17 and Tepe and Williams.18 Lambda
DNA/HindIII was employedas amolecular weight mark-
er (Fig. 2, lane 1). pBR322 DNA cross-linking band was
observed in lane 4, and pUC19 DNA cross-linking band
in lane 5. When mixing pBR322 and pUC19 with com-
pound 5, two DNA cross-linking bands were observed
after illumination (Fig. 2, lane 6). The possible formations
of DNA cross-links between pBR322 and pBR322,
pUC19 and pUC19, and pBR322 and pUC19 were illus-
trated in Scheme 3. If DNA interstrand cross-link hap-
pened only, we could observe two cross-linking bands,
one was 2686 + 2686 (bases), and another was
4363 + 4363 (bases) in the gel. If DNA interstrand
cross-link and interhelical cross-link both occurred, we
would observe three cross-linking bands, 2686 + 2686
cross-linking
bands,
2686 + 2686
(bases) and
4363 + 4363 (bases) (Fig. 2, lane 6), were observed. It sug-
gested that only DNA interstrand cross-link by com-
pound 5 happened (Scheme 3).
Interesting result was found by our investigation for tu-
mor cell apoptosis induced by compound 5. We mixed
both compound 5 and THP-1 cell with or without illu-
mination. Detection of apoptosis in THP-1 cells was car-
ried out by flow cytometry using annexin V-FITC kit,
which was based on the observation that soon after ini-
tiating apoptosis. Most cell types translocated phospha-
tidylserine (PS) from the inner face of the plasma
membrane to the cell surface. Once on the cell surface,
PS could be easily detected by staining with a FITC con-
jugate of annexin V, a protein that has a strong natural
affinity for PS. Externalization of PS occurs earlier than
the nuclear change associated with apoptosis.19e,g While
propidium iodide (PI) dye could stain necrotic cells, it
could not stain viable cells because of the integral cell
membranes (Fig. 3).
1
2
3
4
5
6
Gel origin
DNA X-link
DNA X-link
Apoptosis is a programmed process of cell death accom-
panied by morphological and biochemical features. Tar-
geting apoptotic cell death pathways will provide the
wide-ranging opportunities for the discovery and devel-
opment of novel drugs.19 Most traditional cancer chemo-
therapy drugs show their cytotoxic effects by inducing
apoptosis.20 It was shown that compound 5 could induce
human tumor cell line, THP-1 cells, apoptosis. THP-1
cells with no drug and no irradiation treatment was con-
sidered as control cells (Fig. 3A, 4.9% late apoptosis rate).
Compound 5 with illumination induced strongest cell
apoptosis (Fig. 3D, 62.03% late apoptosis rate). Only
ss DNA (pBR322)
ss DNA (pUC19)
Figure 2. Denaturing alkaline agarose gel (0.9%) for DNA cross-link
by compound 5. Lane 1, Lambda DNA/HindIII (molecular weight
standard, 1.5 lg); lane 2, pBR322 (control, 0.7 lg); lane 3, pUC19
(control, 0.7 lg); lane 4, pBR322 (0.7 lg) + 5 (10 lM) + hm (30 min);
lane 5, pUC19 (0.7 lg) + 5 (10 lM) + hm (30 min); lane 6, pBR322
(0.7 lg) + pUC19 (0.7 lg) + 5 (20 lM) + hm (30 min).

Y. Song et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1660–1664
THP-1 cells control THP-1 cells + hν
1663
A
B
4.12%
4.09%
5.11%
7.62%
21.25%
6.76%
C
D
THP-1 cells + 0.05 mM
compound 5+ hν
THP-1 cells + 0.05 mM
compound 5
1.42%
3.01%
31.4 %
62.03 %
5.69%
1.29%
Figure 3. Tumor cell line THP-1 apoptosis induced by compound 5. The cells were labeled with annexin V-fluorescein isothiocyanate and propidium
iodide (PI). The distribution pattern of live and apoptotic cells was determined by FACS analysis. Viable cells are those with low annexin or no
annexin and PI staining (lower left panel, D3). Early stage apoptotic cells are represented by high annexin and low PI staining (lower right panel, D4).
Later stage apoptotic cells are represented high annexin and high PI staining (upper right panel, D2). And necrosis is represented by cells with high PI
and low annexin staining (upper left panel, D1). (A) only THP-1 cells; (B) THP-1 cells illuminated for 30 min; (C) THP-1 cells treated with 0.05 mM
compound 5; (D) THP-1 cells treated with 0.05 mM compound 5 and illuminated for 30 min.
compound 5 treated cells without illumination or only
illumination treated cells represented weak apoptosis
(Fig. 3B and C, 21.15% and 3.01% late apoptosis rates,
respectively).21 The effect of apoptosis induced by light
might be caused by the plasma membrane slightly dis-
turbed under photoirradiation.19c,19e
References and notes
1. Blackburn, G. M.; Gait, M. J. Nucleic Acids in Chemistry
and Biology; Oxford University Press: New York, 1996.
2. (a) De Silva, I. U.; McHugh, P. J.; Clingen, P. H.; Hartley,
J. A. Nucleic Acids Res. 2002, 30, 3848; (b) Yarnell, A. T.;
Oh, S.; Reinberg, D.; Lippard, S. J. J. Biol. Chem. 2001,
276, 25736; (c) Hartley, J. A.; Hazrati, A.; Kelland, L. R.;
Khanim, R.; Shipman, M.; Suzenet, F.; Walker, L. F.
Angew. Chem., Int. Ed. 2000, 39, 3467; (d) Yarnell, W. S.;
Roberts, J. W. Science (Washington, DC) 1999, 284, 611;
(e) Jamieson, E. R.; Lippard, S. J. Chem. Rev. 1999, 99,
2467.
Effective DNA cross-linking agents might be promising
techniques to design new types of anti-tumor agents.
Our studied indicated that phenol quaternary ammonium
compounds 3, 4, and 5 could inhibit the transcription un-
der illumination. Apparently, compound 5 had a good
shape orientation to adopt helical conformation and
could easy localize on DNA. Therefore, compound 5
had better DNA binding mode and induced DNA inter-
strand cross-link after illumination. This action would
prevent the formation of the ‘open’ structure by holding
two strands together and then inhibit the transcription
and replication process. Furthermore, compound 5 could
induce cell to undergo apoptosis. It will encourage us to
investigate the apoptosis-related drug discovery in future.
3. Reddy, M. C.; Christensen, J.; Vasquez, K. M. Biochem-
istry 2005, 44, 4188.
4. Satz, A. L.; Bruice, T. C. Acc. Chem. Res. 2002, 35, 86.
5. Sorasaenee, K.; Fu, P. K.-L.; Angeles-Boza, A. M.;
Dunbar, K. R.; Turro, C. Inorg. Chem. 2003, 42, 1267.
6. Rajski, S. R.; Williams, R. M. Chem. Rev. 1998, 98, 2723.
7. Zou, Y.; Van Houten, B.; Farrell, N. Biochemistry 1994,
33, 5404.
8. Dunham, S. U.; Chifotodes, H. T.; Mikulski, S.; Burr, A.
E.; Dunbar, K. R. Biochemistry 2005, 44, 996.
9. Kohn, K. W. Cancer Res. 1996, 56, 533.
10. Wang, P.; Liu, R. P.; Wu, X. J.; Ma, H. J.; Cao, X. P.;
Zhou, P.; Zhang, J. Y.; Weng, X. C.; Zhang, X.-L.; Qi, J.;
Zhou, X.; Weng, L. H. J. Am. Chem. Soc. 2003, 125, 1116.
11. (a) Pande, P.; Shearer, J.; Yang, J.; Greenberg, W. A.;
Rokita, S. E. J. Am. Chem. Soc. 1999, 121, 6773; (b)
Wang, P.; Song, Y.; Zhang, L.-X.; He, H.-P.; Zhou, X.
Curr. Med. Chem. 2005, 12(24), 2893.
Acknowledgments
X.Z. thanks the NSFC (National Science Fund of
China) (No. 20272046) and National Science Fund for
Distinguished Young Scholars (No. 20425206) for
financial support.

1664
Y. Song et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1660–1664
12. Chiang, Y.; Kresge, A. J.; Zhu, Y. J. Am. Chem. Soc.
2001, 123, 8089.
bromide, visualized by UV, and photographed using
Vilber Lourmat video system.
13. Zhou, Q.; Rokita, S. E. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 15452.
16. Nakatani, K.; Higashida, N.; Saito, I. Tetrahedron Lett.
1997, 38, 5005.
14. Richter, S. N.; Maggi, S.; Mels, S. C.; Palumbo, M.;
Freccero, M. J. Am. Chem. Soc. 2004, 126, 13973.
15. (a) Fu, P. K.-L.; Turro, C. Chem. Commun. 2001, 279(b)
General protocol for transcription inhibition in vitro. The
transcription experiments were carried out by the T7
control DNA template and the RiboMAX Large-Scale
RNA Production System with T7 RNA Polymerase
(Promega P1300). Various concentrations of drugs mixing
with DNA template were exposed to a 50-W high-pressure
mercury lamp, which was placed 20 cm away at room
temperature. Then T7 reaction components were added
according to transcription procedure (Promega P1300).
The transcription was proceeded at 37 °C for 1 h in
nuclease-free water (HEPES–KOH, pH 7.5) in the pres-
ence of 18 mM MgCl2, 1.5 mM spermidine, 30 mM DTT,
and 3 mM each ATP, CTP, GTP, and UTP. The
transcription mRNA mixtures were loaded onto a native
0.9% agarose gel. The gels were stained by ethidium
17. Cech, T. R. Biochemistry 1981, 20, 1431.
18. Tepe, J. J.; Williams, R. M. J. Am. Chem. Soc. 1999,
121, 2951.
19. (a) Kawanishi, S.; Hiraku, Y. Curr. Med. Chem.—Anti-
Cancer Agents 2004, 4, 415; (b) Nesterenko, V.; Putt, K. S.;
Hergenrother, P. J. J. Am. Chem. Soc. 2003, 125, 14672; (c)
Lawen, A. BioEssays 2003, 25, 888; (d) Oleinick, N. L.;
Morris, R. L.; Belichenko, I. Photochem. Photobiol. Sci.
2002, 1, 1; (e) D’Agnillo, F.; Alayash, A. I. Blood 2001, 98,
3315; (f) Hengartner, M. O. Nature 2000, 407, 770; (g)
Fadok, V. A.; Bratton, D. L.; Frasch, S. C.; Warner, M. L.;
Henson, P. M. Cell Death Differ. 1998, 5, 551.
20. (a) Alam, J. A. Trends Biotechnol. 2003, 21, 479; (b)
Kaufman, S. H.; Earnshaw, W. C. Exp. Cell Res. 2000,
256, 42.
21. Reddy, R. K.; Mao, C.; Baumeister, P.; Austin, R. C.;
Kaufman, R. J.; Lee, A. S. J. Biol. Chem. 2003,
278, 20915.