Action of a novel pyrrolo[1,2-c][1.3]benzodiazepine on the viability of Jurkat and neuronal/glial cells

Article abstract of DOI:10.1016/j.bmcl.2005.05.006

We have designed and synthesized several structural isomers of anthramycin (heterocycles 2, 3, 5, 6, and 8) and found that, in particular, pyrrolobenzodiazepine 8 induces DNA cleavage and formation of small fragments of DNA. The cytotoxic effects of 8 wer

Full text of DOI:10.1016/j.bmcl.2005.05.006

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3220–3223
Action of a novel pyrrolo[1,2-c][1.3]benzodiazepine
on the viability of Jurkat and neuronal/glial cells
Georgios Rotas,a Ketevan Natchkebia,b Nino Natsvlishvili,b Merab Kekelidze,b
Athanasios Kimbaris,a, George Varvounisa,* and David Mikeladzeb,*
aDepartment of Chemistry, University of Ioannina, GR-451 10 Ioannina, Greece
bInstitute of Physiology Georgian Academy of Sciences, Tbilisi, 380003, Georgia
Received 9 March 2005; revised 22 April 2005; accepted 2 May 2005
Available online 31 May 2005
Abstract—We have designed and synthesized several structural isomers of anthramycin (heterocycles 2, 3, 5, 6, and 8) and found
that, in particular, pyrrolobenzodiazepine 8 induces DNA cleavage and formation of small fragments of DNA. The cytotoxic effects
of 8 were manifested with both non-transformed primary neuronal/glial cells and transformed Jurkat cells. The other compounds
did not change the viability either of transformed or of non-transformed cells, and induced DNA cleavage to a lesser extent.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Interactions between the drug molecules and the gua-
nine N2 of DNA are becoming important aspects in
the design of new antitumor compounds. Many natural
antibiotics, including anthramycin,1 daunorobicin and
doxorubicin,2 and ecteinascidins,3 are shown to bind
to the N2 of guanine in the minor groove of DNA.
Anthramycin, an antitumor alkylating antibiotic pro-
duced by Streptomyces refuineus, covalently binds to
the N2 amine of the guanine 9 residues through its
C11 position.4 The cytostatic and antitumor effects of
anthramycin and related benzodiazepines are believed
to be due to their selective interaction with DNA,5
which causes inhibition of nucleic acid synthesis,6 and
production of excision-dependent single- and double-
strand breaks in cellular DNA.7 The DNA adduct in-
duced by anthramycin causes only minimal distortion
of the DNA helix and is poorly recognized and, hence,
poorly removed by the repair proteins.
like other DNA-alkylating antibiotics it induces apopto-
sis by a mitochondrial pathway.8
Another aspect of pyrrolobenzodiazepine activity is the
inhibition of molecular interactions between DNA and
transcription factor Sp1.9 Inhibition of DNA binding
activity of Sp1 to the cognate ÔG–CÕ box causes neuro-
protection against oxidative stress induced by various
excytotoxic stimuli.10 Because of the many side effects
of anthramycin antibiotics (e.g., nausea, vomiting, diar-
rhea, myelosuppression, and a dose-limiting cardiotox-
icity), there have been numerous attempts to obtain
improved derivatives with enhanced activity or reduced
side effects. Taking into account the potential neuropro-
tective and anti-carcinogenic effects of anthramycin re-
lated benzodiazepines, we investigated the change in
survival of transformed human leukemic Jurkat and
non-transformed neuronal/glial cells, induced by novel
pyrrole, pyrrolobenzazepine, and pyrrolobenzodiaze-
pine derivatives.
The action of anthramycin on the apoptosis of cancer
cells is not fully understood, but it may be assumed that
The tested heterocycles 2, 3, 5, 6, and 8 were prepared
from a common starting material, namely, (2-nitrophen-
yl)(1-tosyl-1H-pyrrol-2-yl)methanone 111 (Scheme 1).
Compound 1 was reduced by catalytic hydrogenation
over 5% palladium-on-carbon to give the corresponding
amine, which was then detosylated to compound 2 in
aqueous 2 N NaOH. Compound 2 was either further
Keywords: Pyrrolobenzodiazepine; Neuronal/glial cells; Transformed
Jurkat cells; DNA cleavage.
*
Corresponding authors. Tel.: +30 265198382; fax: +30 265198799
(G.V.); tel.: +99 532 374724; fax: +99 532 941045 (D.M.); e-mail
addresses: gvarvoun@cc.uoi.gr, d.mikeladze@mymail.ge
Present address: Democritius University of Thrace, School of
Agricultural Development, Athanasiou Pantazidou 193, GR-628 00
Orestiada, Greece.
reduced to 2-(1H-pyrrol-2-yl)methyl)benzenamine
with sodium borohydride in refluxing propan-2-ol, or
3
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.006

G. Rotas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3220–3223
3221
Table 1. Effects of 1 lM heterocycles 2, 3, 5, 6 and 8 on the reduction
of MTT in glial/neuronal cells
Additions
MTT reduction (O.D. 570 nm)a
Control
2
0.56 0.08
0.65 0.07
0.38 0.08
0.55 0.09
0.62 0.06
0.58 0.01
0.29 0.05
0.30 0.07
0.22 0.04
0.25 0.07
0.28 0.06
0.17 0.03
3
5
6
8
+ NMDA
+ NMDA + 2
+ NMDA + 3
+ NMDA + 5
+ NMDA + 6
+ NMDA + 8
a Cell viability after exposure to 100 lM NMDA. The data are pre-
sented as means SEM for triplicate determination.
several cytokines in response to stimuli. It is a well-
established model for the study of apoptotic death path-
ways of cancer cells.15 Therefore, in the next series of
experiments, Jurkat cells14 were incubated in the pres-
ence of heterocycles 2, 3, 5, 6, and 8 (Table 2). It was
found that MTT uptake was significantly decreased only
in the presence of compound 8, suggesting that this
derivative has anti-proliferative and pro-apoptotic
activity.
Scheme 1. Reagents and conditions: (i) (a) H2, 5% Pd–C, MeOH, rt,
8 h, (b) aq 2 N NaOH, MeOH, reflux, 5 h; (ii) NaBH4, IPA, reflux,
24 h; (iii) CSCl2, toluene, Et3N, rt, 1 h; (iv) AlCl3, MeNO2, rt, 6.5 h;
(v) K2CO3, DMF, rt, 3 h; (vi) CSCl2, toluene, Et3N, ꢀ45 ꢁC, 5 min.
transformed into the isothiocyanate 4 by reaction with
thiophosgene. Amine 3 was converted into isothiocya-
nate 7 similarly. When 4 was subjected to AlCl3 in nitro-
methane, ring closure via intramolecular electrophilic
substitution between C-3 of the pyrrole ring and isothi-
ocyanate group afforded pyrrolo[3,2-c][1]benzazepine-
10(1H)-one-4(5H)-thione 5. On the other hand, treat-
ment of 4 and 7 with potassium carbonate in DMF
afforded pyrrolo[1,2-c][1.3]benzodiazepines 6 and 8, by
intramolecular nucleophilic addition of the generated
pyrrolyl anion to the isothiocyanate group.12
We have also analyzed DNA strand breaks in cells treat-
ed with 1 lM each of heterocycles 2, 3, 5, 6, and 8 using
pulsed field gel electrophoresis16 (Fig. 1). The additional
DNA fragments of 3–4 Mb appeared in Jurkat cells fol-
lowing a 20 h treatment only with tricycle 8, suggesting
strongly that this compound induced DNA cleavage.
Apparently, in this case DNA cleavage is initiated by
heterocycle 8 mediated inhibition of topoisomerases that
results in provoking poly(ADP)-ribose polymerase acti-
vation and apoptosis. Other compounds cause the form-
ation of DNA fragments to a lesser extent.
N-Methyl-D-aspartate (NMDA) receptor-mediated cell
death is complex, probably involving elements of apop-
tosis in neuronal cells. The cytotoxic effects are mediated
by increased Ca2+ influx through activated NMDA
receptor. Associated with Ca2+ influx is an increase in
reactive oxygen species (ROS) that appears to originate
in the mitochondria. Ca2+ overloading reduces the mem-
brane potential and disrupts electron transport, result-
ing in the increased production of ROS.13 Some types
of DNA-binding drugs through transcription factors
Sp1 inhibit apoptosis and DNA damage in rat cortical
neurons caused by excitotoxic oxidative stress.10 Hence,
the effects of heterocycles 2, 3, 5, 6, and 8 on NMDA-
receptor mediated cell death were examined.14 It was
found that none of the examined compounds have any
protective properties against NMDA-induced excitotox-
icity. In the presence of these compounds, the reduction
rate of MTT did not change (Table 1). Moreover, com-
pound 8 increased cell damage after treatment of cells by
NMDA, suggesting that this compound intensifies
NMDA-dependent excitotoxicity.
The data presented point out that compound 8 causes
cell death in both transformed Jurkat and non-trans-
formed primary neuronal/glial cells, apparently by
inducing damage to DNA. However, the mechanism
of action of this pyrrolobenzodiazepine derivative differs
from anthramycin and other alkylating minor groove
binders, since compound 8 does not contain the imine
or carbinolamine groups that would react with amines
to form covalent bonds. It is suggested that compound
Table 2. Effects of 1 lM heterocycles 2, 3, 5, 6, and 8 on the reduction
of MTT in Jurkat cells
Additions
MTT reduction (O.D. 570 nm)a
Control
0.47 0.05
0.39 0.07
0.34 0.03
0.45 0.07
0.41 0.08
0.22 0.05
2
3
5
6
8
a Cell viability after exposure for 24 h assessed by the MTT tests. The
data are presented as means SEM for triplicate determination.
Jurkat cells are a human leukemic cell line, which
expresses CD4 clusters and has the ability to produce

3222
G. Rotas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3220–3223
6. Kohn, K. W. Anthramycin. In Antibiotics III. Mechanism
of Action of Antimicrobial and Antitumour Agents; Corc-
oran, J. W., Hahn, F. E., Eds.; Springer: New York, 1975,
pp 3–11.
7. Petrusek, R. L.; Uhlenhopp, E. L.; Duteau, N.; Hurley, L.
H. J. Biol. Chem. 1982, 257, 6207.
8. Tada-Oikawa, S.; Kawanishi, M.; Yamada, M.; Kawani-
shi, S. FEBS Lett. 1999, 442, 65.
9. Baraldi, P. G.; Cacciari, B.; Guiotto, A.; Romagnoli, R.;
Spalluto, G.; Leoni, A.; Bianchi, N.; Feriotto, G.; Ruti-
gliano, C.; Mischiati, C.; Gambari, R. Nucleosides Nucle-
otides Nucleic Acids 2000, 19, 1219.
10. Chatterjee, S.; Zaman, K.; Ryu, H.; Conforto, A.; Ratan,
R. R. Ann. Neurol. 2001, 49, 345.
11. Kimbaris, A.; Varvounis, G. Tetrahedron 2000, 56, 9675.
12. (a) Data for 7: as an oil; IR (Neat) mmax 3388, 3097, 3068,
2915, 2105, 1597, 1578 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
dH (ppm) 4.06 (s, 2H, CH2), 6.04 (m, 1H, H-3), 6.19 (m,
1H, H-4), 6.73 (m, 1H, H-5), 7.14–7.26 (m, 4H, H-30, H-40,
H-50, H-60), 7.99 (br s, 1H, NH pyrrole); 13C NMR
(CDCl3, 100 MHz) dC (ppm) 31.26, 107.75, 109.39, 118.03,
127.40, 128.37, 128.43, 129.07, 130.65, 130.77, 135.20,
137.02; MS (EI), m/z 214 (M+, 100), 186 (28), 181 (31), 154
(12), 80 (8); EI HRMS M+, found 214.0559 C12H10N2S
requires 214.0565; (b) Data for 8: as a light yellow powder
mp = 183 ꢁC (dec); IR (Nujol) mmax 3165, 3111, 1609,
1583 cmꢀ1; 1H NMR (CDCl3, 400 MHz) dH (ppm) 3.95 (s,
2H, CH2), 6.03 (m, 1H, H-1), 6.20 (t, 1H, J = 3.3 Hz, H-2),
7.15 (m, 1H, H-10), 7.20–7.29 (m, 3H, H-7, H-8, H-9), 7.68
(dd, 1H, J = 3.4, 1.9 Hz, H-3), 9.91 (br s, 1H, NH); 13C
NMR (CDCl3, 100 MHz) dC (ppm) 31.57, 109.32, 112.26,
120.88, 125.61, 127.28, 127.53, 128.03, 132.58, 132.89,
136.40, 180.17; MS (EI), m/z 214 (M+, 100), 186 (33), 181
(32), 149 (36), 135 (20), 97 (15%); EI HRMS M+, found
214.0556 C12H10N2S requires 214.0565; (c) Compounds 4–
6 were characterized by satisfactory elemental analyses,
Figure 1. Detection of intracellular DNA cleavage in Jurkat cells
treated with heterocycles 2, 3, 5, 6, and 8. Cells (1 · 106/ml were treated
with 1 lM of each heterocycle 2, 3, 5, 6, and 8 at 37 ꢁC for 20 h. Cells,
prepared as agarose plugs, were lysed and subjected to pulsed field gel
electrophoresis through a 1% agarose gel. C, control; M, size marker
DNA.
8 induces genotoxicity by stabilizing the DNA helix just
like the sequence-selective pyrrolobenzodiazepine dimer
SJG-136. The latter is unable to interact covalently with
DNA but has significant cytotoxicity in some cell lines.17
Further experiments are needed for the clarification of
the mechanism of action of non-covalent DNA-interac-
tive pyrrolobenzodiazepine derivatives. The results pre-
sented augment the repertoire of compounds that have
pro-apoptotic properties and may be used as a basis
for the design of new non-toxic anti-cancer drugs.
1
and from their mass, H and 13C NMR spectra.
13. Nichols, D.; Budd, S. Physiol. Rev. 2000, 80, 315.
14. (a) Cerebrocortical cultures—Cortical cultures of mixed
neurons and glia were derived from embryonic (fetal day
15 or 16) Wistar rats. Briefly, following dissociation in
0.027% trypsin, cerebral cortical cells were plated either on
96-well multiwell plates or 35 mm dishes that had been
previously coated overnight with 15 lg/ml poly-L-lysine
and then with DMEM (Sigma) culture medium supple-
mented with 10% fetal bovine serum. After removal of the
final coating solution, cells were seeded (106 ml) in a serum
free medium composed of a mixture of DMEM, contained
40 lg/ml gentamycin, and 60 lg/ml penicillin. Cells were
cultured at 37 ꢁC in a humidified atmosphere of 95% air
and 5% CO2 and were used after 6–7 days; (b) Jurkat cell
culture—The human Jurkat T cell line (clone E6-1) was
obtained from the American Type Culture Collection
(Manassas, VA). Cells were maintained in RPMI 1640
medium (Sigma) supplemented with 10% FBS (Sigma),
2 mM L-glutamine (Life Technologies), and 10 lg/ml
gentamycin (Life Technologies). Cells were grown at
37 ꢁC in a humidified atmosphere consisting of 5% CO2.
Cells were passaged three times weekly and maintained at
a density between 0.2 and 1 · 106 cells/ml. Cells used for
all experiments were in logarithmic growth phase, and the
medium used for experiments had the same constituents as
that used for cell passage, unless otherwise indicated; (c)
MTT reduction cell viability assay—Experiments were
performed using glia/neurons and Jurkat cells cultured in
96-well plates. The effects of heterocycles 2, 3, 5, 6 and 8
(1 lM) or/and NMDA (10 lM) on cell viability was
assessed by a colorimetric assay based on the cleavage of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
Acknowledgments
This work was supported by a grant within the frame-
work of Joint Research and Technology Projects be-
tween Greece (Grant no. 1476/06-02-03) from the
General Secretariat of Research and Technology, Ath-
ens (to G.V.), and Georgia from the State Department
for Science and Technology, Tbilisi (to D.M.). We are
particularly grateful to A. Cakebread and R. Tye for
mass spectra obtained on machines funded by the Uni-
versity of London Intercollegiate Research Services
Scheme.
References and notes
1. (a) Petrusek, R. L.; Anderson, G. L.; Garner, T. F.;
Fannin, Q. L.; Kaplan, D. J.; Zimmer, S. G.; Hurley, L.
H. Biochemistry 1981, 20, 1111; (b) Warpehoski, M. A.;
Hurley, L. H. Chem. Res. Toxicol. 1988, 1, 315.
2. Zhang, H.; Gao, Y.-G.; van der Marel, G. A.; van Boom,
J. H.; Wang, A. H.-J. J. Biol. Chem. 1993, 268, 10095.
3. Sakai, R.; Rinehart, K. L.; Guan, Y.; Wang, A. H.-J.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11456.
4. Yang, X.-L.; Wang, A. H.-J. Pharm. Ther. 1999, 83, 181.
5. Kohn, K. W.; Glaubiger, D.; Spears, C. L. Biochim.
Biophys. Acta 1974, 361, 288.

G. Rotas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3220–3223
3223
mide (MTT) into a blue-colored formazan product by
mitochondrial succinate dehydrogenase. Additions were
made directly to the culture medium for 24 h. Cells then
washed twice with HEPES-buffered incubation medium
(HBM) (140 mM NaCl, 5 mM KCl, 5 mM NaHCO3,
1.1 mM MgCl2, 1.2 CaCl2, 5.5 mM glucose, and 20 mM
HEPES, pH 7.4.) and incubated for 45 min at 37 ꢁC in
HBM containing MTT (0.5 mg/ml). After this period, the
HBM was carefully removed, and the blue formazan
product was solubilized in 300 ll of 100% dimethyl
sulfoxide. The absorbance of each well was read at
570 nm. The data were treated by one-way ANOVA
analysis. The data from each experiment were analyzed
separately. Where a significant effect was observed in the
ANOVA analysis, comparisons of those samples were
made by the t-test.
15. Mohagheghpour, N.; Damle, N. K.; Moonka, D. K.;
Terrell, C. P.; Engleman, E. G. J. Immunol. 1984, 133, 133.
16. Detection of cellular DNA cleavage induced by pulsed field
electrophoresis—Following treatment of heterocycles 2, 3,
5, 6 and 8 with 0.2 lM, cells were washed twice with
phosphate buffered saline (PBS). Cell suspensions were
solidified with agarose, followed by treatment with pro-
teinase K.18 Electrophoresis was performed at 14 ꢁC using
a CHEF-DRII pulsed field electrophoresis system (Bio-
Rad) at 200 V. Switch time was 60 s for 15 h followed by
90 s for 9 h.
17. Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N.
A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston,
D. E. J. Med. Chem. 2001, 44, 737.
18. Ito, K.; Yamamoto, K.; Kawanishi, S. Biochemistry 1992,
31, 11606.