Biologically active carbazole derivatives: Focus on oxazinocarbazoles and related compounds

Article abstract of DOI:10.3109/14756366.2014.899594

Four series of carbazole derivatives, including N-substituted-hydroxycarbazoles, oxazinocarbazoles, isoxazolocarbazolequinones, and pyridocarbazolequinones, were studied using diverse biological test methods such as a CE-based assay for CK2 activity measurement, a cytotoxicity assay with IPC-81 cell line, determination of MIC of carbazole derivatives as antibacterial agents, a Plasmodium falciparum susceptibility assay, and an ABCG2-mediated mitoxantrone assay. Two oxazinocarbazoles Ib and Ig showed CK2 inhibition with IC50=8.7 and 14.0μM, respectively. Further chemical syntheses were realized and the 7-isopropyl oxazinocarbazole derivative 2 displayed a stronger activity against CK2 (IC50=1.40μM). Oxazinocarbazoles Ib, Ig, and 2 were then tested against IPC-81 leukemia cells and showed the ability to induce leukemia cell death with IC50 values between 57 and 62μM. Further investigations were also reported on antibacterial and antiplasmodial activities. No significant inhibitory activity on ABCG2 efflux pump was detected.

Full text of DOI:10.3109/14756366.2014.899594

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2015; 30(2): 180–188
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.899594
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RESEARCH ARTICLE
Biologically active carbazole derivatives: focus on oxazinocarbazoles
and related compounds
Zouhair Bouaziz1, Samar Issa2, Jacques Gentili1, Andreas Gratz3, Andre Bollacke3, Matthias Kassack4, Joachim
5
5
7
7
Jose3,4, Lars Herfindal5,6, Gro Gausdal , Stein Ove Døskeland , Catherine Mullie , Pascal Sonnet , Camille
´
Desgrouas8, Nicolas Taudon8, Glaucio Valdameri9,10, Attilio Di Pietro9, Milad Baitiche11, and Marc Le Borgne1
1
2
EA 4446 Biomolecules Cancer et Chimioresistances, Faculte de Pharmacie - ISPB, Universite Lyon 1, Lyon, France, Ecole de Biologie Industrielle,
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3
4
EBInnov, Cergy-Pontoise, France, IPMC, PharmaCampus, WWU Mu¨nster, Germany, IPMC, Heinrich-Heine-Universita¨t Du¨sseldorf, Germany,
5Department of Biomedicine, University of Bergen, Norway, Translational Signaling Group, Haukeland University Hospital, Bergen, Norway,
6
7
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Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources (LG2A), FRE-CNRS 3517, Universite de Picardie Jules Verne, Amiens, France,
8
9
UMR-MD3, Faculte de pharmacie, Aix-Marseille-Universite, Marseille, France, Drug Resistance Mechanism and Modulation Group, Bases
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Moleculaires et Structurales des Systemes Infectieux (BMSSI), UMR 5086, CNRS, Universite Lyon 1, IBCP, Lyon, France, 10Department of Biochemistry
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11
and Molecular Biology, UFPR, Curitiba, Brazil, and Faculte de Technologie, Universite de Setif-1, Algerie
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Abstract
Keywords
Four series of carbazole derivatives, including N-substituted-hydroxycarbazoles, oxazinocarba-
zoles, isoxazolocarbazolequinones, and pyridocarbazolequinones, were studied using diverse
biological test methods such as a CE-based assay for CK2 activity measurement, a cytotoxicity
assay with IPC-81 cell line, determination of MIC of carbazole derivatives as antibacterial agents,
a Plasmodium falciparum susceptibility assay, and an ABCG2-mediated mitoxantrone assay. Two
oxazinocarbazoles Ib and Ig showed CK2 inhibition with IC50 ¼ 8.7 and 14.0 mM, respectively.
Further chemical syntheses were realized and the 7-isopropyl oxazinocarbazole derivative 2
displayed a stronger activity against CK2 (IC50 ¼ 1.40 mM). Oxazinocarbazoles Ib, Ig, and 2 were
then tested against IPC-81 leukemia cells and showed the ability to induce leukemia cell death
with IC50 values between 57 and 62 mM. Further investigations were also reported on
antibacterial and antiplasmodial activities. No significant inhibitory activity on ABCG2 efflux
pump was detected.
Biological activities, carbazoles, chemical
synthesis, oxazinocarbazoles, screening
History
Received 16 January 2014
Revised 24 February 2014
Accepted 24 February 2014
Published online 2 April 2014
Introduction
a constitutively active serine/threonine protein kinase that is
involved in many diseases including different types of cancer13.
Furthermore, the challenges in cancer and infectious diseases are
to find active compounds that encounter multidrug resistance
frequently associated with overexpression of the ABC transporter
family. Indolocarbazole derivatives represent novel classes of
ABCG2 inhibitors14. Miscellaneous activities for carbazole
derivatives have been reported such as anti-oxidative, anti-
Extensive research in the carbazole chemistry has been reported
since the description of the parent 9H-carbazole in 1872, by
Graebe and Glaser1. The discovery of the antibacterial activity
of murrayanine extracted from the stem bark of Murraya Koenigii
(Rutaceae) led to the discovery of new pharmacological
activities2,3. Wu et al.4 described the antiplatelet and vasorelaxing
activities of carbazole derivatives extracted from the Clausena
excavata and recently, Peng et al.5 described new derivatives
excavatine B and excavatine C with cytotoxic activity and
antimicrobial activities. Many examples of compounds such as
5H-benzo[b]carbazoles6, furo[3,4-b]carbazoles7, pyrrolo[2,3-
a]carbazoles8,9 as well as indolocarbazoles10 have shown
antitumor activities. Nowadays, targeted therapies have become
one of the main research activities and kinases have reached the
second most important drug target after G-coupled protein
receptors11. Prudent et al.12 have shown the activity of
pyridocarbazole as casein kinase 2 (CK2) inhibitors; CK2 is
inflammatory15, and antibacterial activities16,17
.
To our knowledge, little has been described on the biological
activity of oxazinocarbazole derivatives18,19. We have described
the synthesis and cytotoxic activities of oxazinocarbazoles20.
In this report, we focus on a large panel of pharmaceutical
applications ranging from antibacterial, antimalarial activities as
well as CK2 and ABCG2 inhibition enriched with the synthesis
and activity of new carbazole derivatives.
Materials and methods
Chemistry
Reagents were commercial products of the highest available
purity grade. Mitoxantrone was purchased from Sigma Aldrich
(Lyon, France). Melting points were determined on an
Electrothermal 9200 capillary apparatus (Wolf Laboratories
Address for correspondence: Marc Le Borgne, PhD, EA 4446
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Biomolecules Cancer et Chimioresistances, Faculte de Pharmacie -
ISPB, Universite Lyon 1, Lyon, France. E-mail: marc.le-borgne@
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´
´
univ-lyon1.fr

DOI: 10.3109/14756366.2014.899594
Biologically active carbazole derivatives 181
Limited, Pocklington, UK). The IR spectra were recorded on a 30 min at 0 ꢁC. A solution containing 1 mmol of hydroxycarbazole
Perkin Elmer Spectrum Two IR Spectrometer (Perkin Elmer, 1 dissolved in 10 mL of methanol was added dropwise. The
Waltham, MA). 1H NMR and 13C NMR spectra (broadband mixture was then stirred at room temperature for 3 h to give a
decoupling and DEPT-135) were recorded on a Brucker Avance precipitate of the aminometylated compound which was purified
400 (400 MHz for 1H and 133 MHz for 13C) or a Brucker Avance by column chromatography on silica gel (CH2Cl2/petroleum
500 spectrometer (Perkin Elmer, Waltham, MA) (500 MHz for 1H ether: 2/1).
and 126 MHz for 13C) using CDCl3 or DMSO-d6 as solvents.
Chemical shifts (ꢀ) are referred to that of the solvent. Low 3-[(Benzylmethylamino)methyl]-9-isopropyl-9H-carbazol-4-ol (3)
resolution mass spectra were recorded on an Agilent 1290 Infinity
1
Yield 67%; mp ¼ 150 ꢁC; IR: 2836, 1636, 1601 cmꢀ1; H NMR
system (Agilent technologies, Palo Alto, CA) equipped with an
(ꢀ, CDCl3): 8.39 (d, 1H, J ¼ 7.7 Hz, Harom.); 7.39 (d, 1H,
Agilent 1260 DAD detector and an Agilent 6120 Quadrupole
J ¼ 8.3 Hz, Harom.); 7.33–7.13 (m, 8H, 7Harom. and OH); 6.96
mass detector with an ESI source in a positive mode. HRMS
(d, 1H, J ¼ 8.3 Hz, Harom.); 6.85 (d, 1H, J ¼ 8.3 Hz, Harom.);
spectra were performed on a Q-TOF Micro Water with an ESI
4.85 (m, 1H, CHMe2); 3.84 (s, 2H, CH2); 3.58 (s, 2H, CH2); 2.21
source in the positive mode. Flash chromatography was per-
(s, 3H, NMe); 1.60 (d, 6H, J ¼ 7.1 Hz, CHMe2); 13C NMR (ꢀ,
formed on 230–400-mesh silica.
CDCl3): 154.8 (1Cq); 141.2 (1Cq); 139.4 (1Cq); 137.4 (1Cq);
129.8 (2CH); 128.9 (2CH); 127.9 (1CH); 126.1 (1CH); 124.6
(1CH); 123.7 (1CH); 123.3 (1Cq); 119.0 (1CH); 112.4 (1Cq);
110.9 (1Cq); 109.5 (1CH); 100.9 (1CH); 61.7 (1CH2); 61.4
(1CH2); 47.0 (1CH); 41.5 (1CH3); 21.1 (2CH3); HRMS calculated
for C24H27N2O, MH+ ¼ 359.2118, found, MH+ ¼ 359.2120.
Synthesis of 9-isopropyl-9H-carbazol-4-ol (1)
In a dried tricol containing 263 mg of NaH 60% (18.75 mmol)
under an argon atmosphere, 500 mg of 4-hydroxycarbazole
(2.73 mmol) dissolved in 0.4 mL of DMF and 5 mL of anhydrous
THF were added dropwise. Within 10 min, 0.3 mL of 2-
iodopropane (3 mmol) was added. The mixture was kept at
room temperature and under an argon atmosphere for 2 h. The
residue was hydrolyzed and neutralized with 15% HCl. After
extraction with CH2Cl2, the organic phase was dried over Na2SO4
and concentrated under vacuum. The residue was purified by
column chromatography on silica gel (CH2Cl2/Petroleum ether: 1/
3-{[(2-Chlorobenzyl)methylamino]methyl}-9-isopropyl-9H-car-
bazol-4-ol (4)
Yield 63%; mp ¼ 61 ꢁC; IR: 2925, 1636, 1603 cmꢀ1; 1H NMR (ꢀ,
CDCl3): 8.44 (d, 1H, J ¼ 7.7 Hz, Harom.); 7.48–7.19 (m, 8H,
7Harom. and OH); 7.06 (d, 1H, J ¼ 8.3 Hz, Harom.); 6.94 (d, 1H,
J ¼ 8.3 Hz, Harom.); 4.93 (m, 1H, CHMe2); 3.98 (s, 2H, CH2);
3.83 (s, 2H, CH2); 2.32 (s, 3H, NMe), 1.69 (d, 7.0 Hz, 6H,
CHMe2); 13C NMR (ꢀ, CDCl3): 154.6 (2Cq); 141.2 (1Cq); 139.3
(1Cq); 135.2 (2Cq); 131.9 (1CH); 130.2 (1Cq); 129.3 (1CH);
127.3 (1CH); 126.2 (1CH); 124.6 (1CH); 123.7 (1CH); 123.2
(1Cq); 119.0 (1CH); 110.9 (1Cq); 109.5 (1CH); 100.9 (1CH);
61.5 (1CH2); 59.1 (1CH2); 47.0 (1CH3); 41.4 (1CH); 21.1 (2CH3);
HRMS calculated for C24H26ClN2O, MH+ ¼ 393.1728, found,
MH+ ¼ 393.1728.
1). Yield 65%; viscous oil; IR: 3450, 1631, 1599, 1580 cmꢀ1; H
1
NMR (ꢀ, CDCl3): 8.36 (m, 1H, Harom.); 7.53 (d, 1H, J ¼ 8.3 Hz,
Harom.); 7.44 (m, 1H, Harom.); 7.30–7.24 (m, 2H, Harom.); 7.13
(d, 1H, J ¼ 8.3 Hz, Harom.); 6.56 (dd, 1H, J1 ¼ 7.8 Hz,
J2 ¼ 0.6 Hz, Harom.); 5.41 (s, 1H, OH); 4.99 (m, 1H, CHMe2);
1.72 (d, 6H, J ¼ 7.1 Hz, CHMe2); 13C NMR (ꢀ, CDCl3): 152.4
(1Cq); 141.8 (1Cq); 139.2 (1Cq); 126.2 (1CH); 124.9 (1CH);
123.3 (1CH); 122.7 (1Cq); 119.2 (1CH); 111.8 (1Cq); 109.9
(1CH); 104.7 (1CH); 103.2 (1CH); 47.2 (1CH); 21.1 (2CH3);
HRMS calculated for C15H15NNaO, MNa+ ¼ 248.1046, found,
MNa+ ¼ 248.1046.
3-{[(3-Chlorobenzyl)methylamino]methyl}-9-isopropyl-9H-car-
bazol-4-ol (5)
1
Yield 47%; mp ¼ 182 ꢁC; IR: 2967, 2833, 1633, 1598 cmꢀ1; H
Synthesis of 7-isopropyl-3-phenyl-2,3,4,7-tetrahydro[1,3]oxa-
zino[5,6-c]carbazole (2)
NMR (ꢀ, CDCl3): 8.46 (d, 1H, J ¼ 7.7 Hz, Harom.); 7.48 (d, 1H,
J ¼ 8.3 Hz, Harom.); 7.42–7.21 (m, 7H, 6Harom. and OH); 7.05
(d, 1H, J ¼ 8.3 Hz, Harom.); 6.95 (d, 1H, J ¼ 8.3 Hz, Harom.);
4.99 (m, 1H, CHMe2); 3.94 (s, 2H, CH2); 3.64 (s, 2H, CH2); 2.31
(s, 3H, NMe), 1.69 (d, 6H, J ¼ 7.0 Hz, CHMe2); 13C NMR (ꢀ,
CDCl3): 154.6 (1Cq); 141.3 (1Cq); 139.5 (1Cq); 139.4 (1Cq);
134.6 (1Cq); 130.3 (1CH); 129.9 (1CH); 128.1 (1CH); 127.9
(1CH); 126.1 (1CH); 124.7 (1CH); 123.7 (1CH); 123.2 (1Cq);
119.1 (1CH); 112.4 (1Cq); 110.7 (1Cq); 109.6 (1CH); 101.0
(1CH); 61.5 (1CH2); 61.0 (1CH2); 47.0 (1CH3); 41.6 (1CH); 21.1
(2CH3); HRMS calculated for C24H26ClN2O, MH+ ¼ 393.1728,
found, MH+ ¼ 393.1726.
A solution containing 1.5 mmol of aniline and 3 mmol of
formaldehyde dissolved in 10 mL methanol was stirred for
30 min at 0 ꢁC. A solution containing 1 mmol of hydroxycarbazole
1 dissolved in 10 mL of methanol was added dropwise. The
mixture was then stirred at room temperature for 24 h. At the end
of the reaction, the solvent was evaporated under vacuum and the
residue was purified by column chromatography on silica gel
(CH2Cl2/cyclohexane: 1/6). Yield 63%; mp ¼ 132.4 ꢁC; IR: 1627,
1
1601, 1578 cmꢀ1; H NMR (ꢀ, CDCl3): 8.54 (d, 1H, J ¼ 7.7 Hz,
Harom.); 7.67 (d, 1H, J ¼ 8.3 Hz, Harom.); 7.59 (m, 1H, Harom.);
7.47–7.37 (m, 7H, Harom.); 7.12 (m, 1H, Harom.); 5.81 (s, 2H,
H-2); 5.13 (m, 1H, CHMe2); 5.00 (s, 2H, H-4); 1.87 (d, 6H,
J ¼ 7.0 Hz, CHMe2); 13C NMR (ꢀ, CDCl3): 150.9 (1Cq); 149.1
(1Cq); 140.1 (1Cq); 139.2 (1Cq); 129.5 (2CH); 124.8 (1CH);
124.0 (1CH); 123.7 (1CH); 122.5 (1Cq); 121.7 (1CH); 119.0
(3CH); 112.2 (1Cq); 110.0 (1Cq); 109.7 (1CH); 103.1 (1CH);
47.0 (1CH); 80.2 (1CH2); 51.0 (1CH2); 21.1 (2CH3); HRMS
3-{[(4-Chlorobenzyl)methylamino]methyl}-9-isopropyl-9H-car-
bazol-4-ol (6)
Yield 67%; mp ¼ 139 ꢁC; IR: 2985, 2954, 2845, 1633, 1603 cmꢀ1
;
1H NMR (ꢀ, CDCl3): 8.46 (d, 1H, J ¼ 7.9 Hz, Harom.); 7.48 (d,
1H, J ¼ 8.1 Hz, Harom.); 7.43–7.21 (m, 7H, 6Harom. and OH);
7.04 (d, 1H, J ¼ 8.1 Hz, Harom.); 6.95 (d, 1H, J ¼ 8.3 Hz,
Harom.); 4.94 (m, 1H, CHMe2); 3.93 (s, 2H, CH2); 3.63 (s, 2H,
CH2); 2.30 (s, 3H, NMe), 1.69 (d, 6H, J ¼ 7.2 Hz, CHMe2); 13C
NMR (ꢀ, CDCl3): 154.6 (1Cq); 141.3 (1Cq); 139.4 (1Cq); 135.9
(1Cq); 133.7 (1Cq); 131.1 (2CH); 129.0 (2CH); 126.1 (1CH);
124.7 (1CH); 123.6 (1CH); 123.2 (1Cq); 119.1 (1CH); 112.4
(1Cq); 110.7 (1Cq); 109.6 (1CH); 101.0 (1CH); 61.4 (1CH2); 60.8
calculated
for
C23H23N2O,
MH+ ¼ 343.1805,
found,
MH+ ¼ 343.1811.
General procedure for the aminomethylation of 9-isopropyl-9H-
carbazol-4-ol (1)
A solution containing 2 mmol of primary amine and 2 mmol of
formaldehyde dissolved in 10 mL of methanol was stirred for

182 Z. Bouaziz et al.
J Enzyme Inhib Med Chem, 2015; 30(2): 180–188
(1CH2); 47.0 (1CH3); 41.5 (1CH); 21.1 (2CH3); HRMS calculated The separated substrate and product peptide were detected at
for C24H26ClN2O, MH+ ¼ 393.1728, found, MH+ ¼ 393.1727.
3-{3-[(Benzylmethylamino)methyl]-9-isopropyl-9H-carbazol-4-
yloxy}propan-1-ol (7)
214 nm using a DAD-detector. Pure solvent was used as a negative
control (0% inhibition) and assays devoid of CK2 were used as a
positive control (100% inhibition). For primary testing an
inhibitor concentration of 10 mM was used. Compounds that
revealed at least 50% inhibition at 10 mM were used for IC50
determinations. The IC50 inhibition was determined using nine
inhibitor concentrations ranging from 0.001 mM to 100 mM. IC50
values were calculated from the resulting dose–response curves.
A
solution containing 100 mg (0.28 mmol) of 3, 38 mg
(0.28 mmol) of 3-bromopropan-1-ol, 77 mg (0.56 mmol) of
K2CO3 and 10.3 mg (0.028 mmol) of tetrabutylammonium
iodide (TBAI) dissolved in 5 mL of CH3CN was stirred at 80 ꢁC
for 4 h. The mixture was then filtered on celite and washed with
CH3CN. The filtrate was evaporated and the residue was purified
by column chromatography on silica gel (EtOAc/petroleum ether:
2/1) to give 7 as a liquid product. Yield 65%; IR: 3381, 2930,
Cytotoxicity assay
IPC-81 acute myelogenic leukemia rat cells23 were maintained at
logarithmic growth in the medium (DMEM) supplemented with
10% horse serum. For cytotoxicity testing, the IPC-81 cells were
seeded in 96 well tissue culture plates at 150 000 cells/mL and
exposed to various concentrations of CK2 inhibitors or vehicles
for 24 h. The cells were next fixed in 2% buffered formaldehyde
(pH 7.4) with the DNA-specific dye Hoechst 33342 (Polysciences
Inc., Eppelheim, Germany) and scored for death by fluorescence
microscopic evaluation of apoptotic chromatin condensation.
At least 100 cells in three different microscopic fields were
counted. IPC-81 cells were also analyzed for intactness of
metabolic activity (formazan oxidation) by the WST-1 assay
(Roche Diagnostics, Basel, Switzerland) following the supplier’s
instructions.
2875, 1622, 1596 cmꢀ1 1H NMR (ꢀ, CDCl3): 8.19 (d, 1H,
;
J ¼ 7.9 Hz, Harom.); 7.46–7.13 (m, 10H, Harom.); 4.89 (m, 1H,
CHMe2); 4.25 (t, 2H, J ¼ 5.8 Hz, CH2); 3.96 (t, 2H, J ¼ 5.8 Hz,
CH2); 3.72 (s, 2H, CH2); 3.51 (s, 2H, CH2); 3.05 (bs, 1H, OH);
2.17 (s, 3H, NMe); 2.13 (m, 2H, CH2); 1.62 (d, 6H, J ¼ 7.0 Hz,
CHMe2); 13C NMR (ꢀ, CDCl3): 154.0 (1Cq); 141.2 (1Cq); 139.6
(1Cq); 139.0 (1Cq); 129.6 (2CH); 129.5 (1CH); 128.5 (2CH);
127.3 (1CH); 125.3 (1CH); 123.1 (1CH); 121.9 (1Cq); 120.7
(1Cq); 119.3 (1CH); 116.8 (1Cq); 110.1 (1CH); 106.3 (1CH);
71.3 (1CH2); 62.0 (1CH2); 60.6 (1CH2); 57.4 (1CH2); 47.1
(1CH3); 42.8 (1CH); 33.2 (1CH2); 21.1 (2CH3); HRMS calculated
for C27H33N2O2, MH+ ¼ 417.2537, found, MH+ ¼ 417.2531.
Preparation of recombinant human CK2 holoenzyme
Determination of antibacterial activity
The preparation of the human recombinant CK2 holoenzyme was
performed according to the protocol previously described by
Olgen et al.21. The a-subunit (CSNK2A1) and b-subunit
(CSNK2B) of the human protein kinase CK2 were expressed in
a bacterial pT7-7 system in Escherichia coli BL21 (DE3). Newly
transformed starter cultures were grown overnight at 37 ꢁC in LB-
medium until they reached the stationary phase. With the separate
starter cultures, new medium was inoculated for both subunits and
cultivated until an OD500 of 0.6 was reached. Protein expression
was induced by addition of IPTG (1 mM final concentration) and
carried out at 30 ꢁC for 5–6 h for CSNK2A1 and at 37 ꢁC for 3 h
for CSNK2B. Bacterial cells were harvested by centrifugation
(6000 ꢂ g for 10 min at 4 ꢁC) and disrupted by sonification (three
times 30 s on ice). Preparations were then centrifuged to remove
the cell debris and the bacterial extracts for both subunits and
combined before purification by a three-column procedure. The
fractions containing active CK2 holoenzyme were determined by
activity measurement using the synthetic peptide substrate
RRRDDDSDDD. Finally, fractions exhibiting CK2 activity
were combined and analyzed by SDS-PAGE and Western Blot.
The following reference strains were used for testing the
antibacterial activity: E. coli DSM 1103 and Pseudomonas
aeruginosa DSM 1117 for Gram negative bacteria (Deutsche
Sammlung fu¨r Mikroorganismen, Braunschweig, Germany) and
Staphylococcus aureus CIP 103.429 and Enterococcus faecalis
CIP 103.214 (Collection de l’Institut Pasteur, Paris, France) for
Gram-positive bacteria. Bacteria were grown overnight at 35 ꢁC in
Tryptic Soy Broth and streaked on Tryptic Soy Agar (AES, Bruz,
France). From these isolation plates, inocula were prepared
according to CLSI recommendations24. The broth microdilution
technique was carried out as advised using selected carbazole
derivatives dissolved in DMSO at concentrations ranging from
0.0625 to 128 mg/mL. Ciprofloxacin and DMSO were used in
each series of experiments as a positive and a negative control,
respectively. The MIC was determined as the lowest drug
concentration at which wells remained visually clear.
Plasmodium falciparum susceptibility assay
The in vitro activities of oxazinocarbazole derivatives were tested
over a concentration range of 0.78–10 000 nM against the
susceptibility to chloroquine (CQ) strain P. falciparum 3D7.
The traditional labeled hypoxanthine method was used to assess
antimalarial activity, as described by Desjardins and coworkers25.
CQ was routinely included as positive controls as well as negative
controls using a solvent. The resulting IC50s were calculated using
Pk-Fit software26 (Simulations Plus, Inc., Lancaster, CA).
Inhibition growth (I) and corresponding drug concentration (C)
were fitted according a sigmoid model, described as
Capillary electrophoresis-based assay for the testing of
inhibitors of the human CK2
The recently established capillary electrophoresis CK2 activity
assay was used for testing the inhibitors22. Therefore, 2 mL of the
dissolved inhibitors (stock solution in DMSO) were mixed with
78 mL of CK2 supplemented kinase buffer which was composed
of 1 mg CK2 holoenzyme, 50 mM Tris/HCl (pH 7.5), 100 mM
NaCl, 10 mM MgCl2, and 1 mM DTT. The reaction was initiated
by the addition of 120 mL assay buffer, which was composed of
25 mM Tris/HCl (pH 8.5), 150 mM NaCl, 5 mM MgCl2, 1 mM
DTT, 100 mM ATP, and 0.19 mM of the substrate peptide
RRRDDDSDDD. The reaction was carried out for 15 min at
37 ꢁC and stopped by the addition of 4 mL EDTA (0.5 M).
Subsequently the reaction mixture was analyzed by a PA800
capillary electrophoresis from Beckman Coulter (Krefeld,
Germany). Acetic acid (2 M, adjusted with conc. HCl to a pH
of 2.0) was used as the electrolyte for electrophoretic separation.
À
Á
I ¼ ðImax ꢃ CꢁÞ= Cꢁ þ IC5ꢁ0
where Imax is the maximum inhibition growth and gamma the
sigmoid factor of the curb.
Inhibitory activity on human ABCG2-transfected cells
All compounds were dissolved in DMSO and then diluted in the
DMEM high glucose medium. The stock solution was stored at

DOI: 10.3109/14756366.2014.899594
Biologically active carbazole derivatives 183
ꢀ20 ꢁC and warmed to 25 ꢁC just before use. The human
The oxazino[5,6-c]carbazoles Ia–p were prepared by a
fibroblast HEK293 cell line transfected with ABCG2 was obtained Mannich type condensation of N-substituted 4-hydroxycarbazole
as previously described27. Cells were maintained in Dulbecco’s with 1.5 eq of various primary amines and 3 eq of formaldehyde.
modified Eagle’s medium (DMEM high glucose), supplemented The Mannich reaction of N-substituted 2-hydroxycarbazole with
with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, the primary amines and formaldehyde led to a mixture of the two
and drug supplemented with 0.75 mg/mL G418. To perform the regioisomers, the oxazino[6,5-b]carbazoles IIa–c and the oxa-
ABCG2-mediated mitoxantrone assay, HEK293-ABCG2 cells zino[5,6-a]carbazoles IIIa–e which were isolated by chromatog-
were seeded at a density of 1 ꢂ 105 cells/well into 24-well culture raphy. Identification of the regioisomeric oxazinocarbazoles was
1
plates. After 48 h incubation, the cells were exposed to 5 mM unambiguously established from their H NMR 300 MHz spectra
mitoxantrone for 30 min at 37 ꢁC, in the presence or absence of (Figure 6)20. Thus, for oxazinocarbazoles II, the 1H NMR spectra
compounds at various concentrations. The cells were then washed shows two singlets at 7.63 and 6.78 ppm for the aromatic protons
with phosphate buffer saline (PBS) and after being trypsinized H-5 and H-10 while in the regioisomers III, it exhibits two
and subsequently resuspended in ice-cold PBS (0.2 mL), they doublets at 7.83 and 6.78 ppm for the aromatic protons H-5 and
were kept on ice until analysis by flow cytometry. The data of H-6 with 8.5 Hz as a coupling constant value.
intracellular drug fluorescence were monitored with a FACS
The N-isopropylated compound 1 was chemoselectively
Calibur cytometer (Becton Dickinson, Franklin Lakes, NJ) using a prepared by treatment of 4-hydroxycarbazole with sodium
FL4-H channel. At least 10 000 events were collected, for which hydride in the presence of isopropyl iodide in the solvent
the maximal fluorescence (100%) was the difference between system THF-DMF. Then, compound 2 was prepared by a Mannich
geometric mean fluorescence of cells incubated with 5 mM of reaction of 1 with 1.5 eq of aniline and 3 eq of formaldehyde in
GF120918 as a control (100% inhibition) and without a inhibitor. MeOH at room temperature. In contrast, syntheses of 3–6 have
been carried out using a Mannich reaction of 1 with 2.5 eq of the
Results and discussion
corresponding secondary amines and 2.5 eq of formaldehyde in
methanol at room temperature. The O-alkylation of 3 with 1 eq of
3-bromopropan-1-ol in the presence of 0.1 eq of tetrabutylammo-
Chemistry
Among the 43 carbazole derivatives described in this study, nium iodide (TBAI) and 2 eq of anhydrous K2CO3 in CH3CN at
the synthesis of only 2–7 has never been reported until now 80 ꢁC gave the compound 7 (Scheme 1).
(Figure 1).
For the other series, experimental works are previously Biological results
described. The N-substituted hydroxycarbazoles 8 and 9 were
Carbazoles derivatives as potent inhibitors of human protein
prepared in one step by a chemoselective N-alkylation of 2- or
kinase CK2
3-hydroxycarbazole with 2.5 eq of NaH in DMF/THF mixture
under argon atmosphere at room temperature and subsequent Thirty-seven compounds were evaluated by the capillary electro-
treatment with the alkylating agent20,28. The tricyclic carbazole- phoresis (CE)-based assay22 for human protein kinase CK2
quinones 10–12 were obtained starting from the corresponding activity measurement and inhibitor screening (Table 1). With this
29,30
´
hydroxycarbazoles by oxidation with Fremy’s salt (Figure 2)
.
recently developed assay, the produced and purified recombinant
Syntheses of pyridocarbazolediones 13 and 14 have been human CK2 holoenzyme was used. The synthetic peptide
achieved using regiospecific hetero-Diels–Alder reaction between RRRDDDSDDD was used as the substrate, which is reported to
an azadiene and an appropriate bromocarbazolequinone29.
Syntheses of isoxazolocarbazolediones 15 and 16 have been
accomplished through a regioselective 1,3-dipolar cycloaddition
of nitrile oxide with an appropriate bromocarbazolequinone
(Figure 3)31.
R
1
O
OH
R
2
N
The oxazinopyrimidocarboline derivatives 17 and 18 were
synthesized through a Mannich reaction employing 5-hydroxy-
b-carboline, 4 eq of formaldehyde and 2 eq of a primary amine in
MeOH at room temperature (Figure 4)32.
Dimers 19 and 20 were obtained as by-products through a
dimerization process in the course of the Mannich condensation
among N-substituted 4-hydroxycarbazole, formaldehyde, and
2-(aminomethyl)pyridine as the primary amine (Figure 5)20.
N
N
Et
OH
Et
O
8
9
10: R = H, R = H
11: R = H, R = Br
12: R = OH, R = H
1
2
1
2
1
2
Figure 2. Structures of tricyclic hydroxycarbazole and carbazolequinone
derivatives 8–12.
OH
OH
O
CH
3
O
N
N
CH
3
N
N
R
N
N
3: R = H
7
2
4: R = ortho-Cl
5: R = meta-Cl
6: R=para-Cl
Figure 1. Structures of oxazinocarbazole 2 and aminomethylated carbazoles 3–7.

184 Z. Bouaziz et al.
J Enzyme Inhib Med Chem, 2015; 30(2): 180–188
Me
O
O
O
O
O
O
N
Me
N
O
N
N
N
N
N
N
O
N
Me
Et
Et
Et
Et
N
O
O
Me
15
13
14
16
Figure 3. Structures of pyrido and isoxazolocarbazolediones 13–16.
O
N
O
N
N
N
N
N
N
N
17
18
Figure 4. Structures of oxazinopyrimidocarboline derivatives 17 and 18.
Figure 5. Structures of N,N-bis(carbazolyl-
methyl)amines 19 and 20.
N
N
OH
OH
OH
N
OH
N
N
N
Et
N
N
Et
19
20
R
Figure 6. Structures of oxazinocarbazoles
Ia–p, IIa–c, and IIIa–e.
2
O
N
5
R
2
N
N
N
O
10
II
R
R
1
1
I
Ia: R = Me, R = CH CH=CH
IIa: R = Me, R = CH -CH=CH
1 2 2 2
1
2
2
2
Ib: R = Me, R = C H
IIb: R = Me, R = C H
1
2
6
5
1
2
6
5
Ic: R = Me, R = CH C H
1
2
2 6 5
H C
2
N
Id: R = Me, R = CH C H (p-CH )
IIc: R = Me, R =
1
2
2
6
4
3
1
2
6
S
N
H C
2
Ie: R = Me, R =
1
2
5
If: R = Et, R = CH CH=CH
2
1
2
2
Ig: R = Et, R = C H
1
2
6 5
N
O
Ih: R = Et, R = CH C H
1
2
2 6 5
R1
Ii: R = Et, R = CH(CH )C H
1
2
3
6 5
N
Ij: R = CH CH=CH , R = CH CH=CH
III
1
2
2
2
2
2
R2
IIIa: R = Me, R = CH -CH=CH
2
Ik: R = CH CH=CH , R = CH C H
1
2
2
2
2
6
5
H C
N
2
1
2
2
II: R = CH CH=CH , R =
1
2
2
2
IIIb: R = Me, R = CH C H
1
2
2 6 5
IIIc: R = Me, R = CH C H (p-CH )
1
2
2
6
4
3
Im: R = CH CH=C(CH ) , R = C H
6 5
IIId: R = Me, R = CH(CH )C H
1
2
3 2
2
1
2
3
6 5
In: R = CH CH=C(CH ) , R = CH C H
1
2
3 2
2
2 6 5
H C
N
2
Io: R = CH CH=C(CH ) , R = CH C H (p-CH )
1
2
3 2
2
2
6
4
3
IIIe: R = Me, R =
1
2
Ip: R = CH CH=C(CH ) , R = CH(CH )C H
1
2
3 2
2
3
6 5

DOI: 10.3109/14756366.2014.899594
Biologically active carbazole derivatives 185
CH
3
HN
OH
OH
OH
R
I
CH
3
N
NaH / Ar
N
HCHO
N
N
H
R
DMF / THF / rt
EtOH, rt
1 (65%)
3: R = H (67%)
4: R = ortho-Cl (63%)
5: R = meta-Cl (47%)
6: R = para-Cl (67%)
Br
HO
Bu
Bu
Bu
NH
2
N
Bu I
HCHO
K CO / CH CN
2
3
3
MeOH, rt
OH
80°C
O
N
O
CH
3
N
N
N
2 (63%)
7 (65%)
Scheme 1. Synthesis of compounds 1–7.
Table 1. Inhibition of human protein kinase CK2 by carbazole derivatives
10–16, Ia–p, IIa–c, and IIIa–e.
hCK2
Compound
(%) Inhibition at 10 mM
IC50 (mM)
10
11
26
20
–
–
12
13
14
15
16
Ia
Ib
Ic
Id
Ie
19
–
–
–
–
–
–
8.7
–
–
24a,b
Not soluble
18a,b
29a,b
20
52
29
18
26
–
If
Ig
Ih
21
58
14
17
–
14.0
–
Figure 7. SDS-PAGE of purified human protein kinase CK2 holoenzyme.
About 15 mL of purified protein solution (0.25 mg/mL) were separated on a
12.5% acrylamide gel and stained with Coomassie Brilliant Blue G250.
At the left, the apparent molecular mass of the marker proteins (lane 1) is
given. Lane 2 shows the purified human CK2 enzyme holoenzyme
(3.75 mg). The band below the CK2a belongs to the well-known
degradation product of the a-subunit CK2a (amino acids 1–335), which
is supposed to be enzymatically active and using occurs during
Ii
–
Ij
Ik
Il
Im
In
Io
Ip
IIa
IIb
IIc
IIIa
IIIb
IIIc
IIId
IIIe
emodin
09
15
25
26
07
15
34
33
18
15
17
26
25
31
17
99
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.58
purification33
.
be most efficiently phosphorylated by CK2. The purity of the
CK2 holoenzyme was higher than 99% (Figure 7)33. We can
directly monitor the degree of CK2 substrate phosphorylation
without pretreatment of the kinase reaction before capillary
electrophoresis. This enabled us to reliably determine CK2
activity/inhibition.
For initial testing, inhibition was determined relative to the
aAll target compounds were tested at 10 mM, except for compounds 13, control at inhibition concentration of 10 mM of compounds
15, and 16 (tested with saturated solutions appeared to be below 10 mM).
bCompounds were tested in triplicates, except for compounds 13, 15, and
16 (only once experiment).
dissolved in DMSO. Negative control cells were cultured with
pure DMSO that never exceeded 1% as a final concentration and

186 Z. Bouaziz et al.
Table 2. Inhibition of human protein kinase CK2 by
oxazinocarbazole 2 and aminomethylated carbazoles 3–7.
J Enzyme Inhib Med Chem, 2015; 30(2): 180–188
hCK2
Compound
(%) Inhibition at 10 mM
IC50 (mM)
2
3
4
5
6
7
71
26
31
29
32
16
1.401
–
–
–
–
–
Figure 8. The ability of oxazinocarbazoles to induce IPC-81 leukemia
cell death. IPC-81 cells were incubated with various concentrations of the
compounds Ib, Ig, and 2, and apoptosis assessed by microscopic
evaluation of leukemia cell morphology after 24 h.
set to 0% inhibition. Reactions without CK2 were used as the
positive control and set to 100% inhibition. Compounds with
more than 50% inhibition at a concentration of 10 mM were
subjected to an IC50 determination. For this purpose, inhibition
was measured at final concentrations ranging from 0.001 to
100 mM in appropriate intervals. IC50 were calculated from the
resulting dose–response curves. Each value was determined at
least in triplicate in independent experiments22.
Table 3. Antimicrobial activities (MIC mg/mL) of carbazole derivatives
8–10, 12, 17–20, Ic, Ik,l, Ip, and IIc.
MIC (mg/mL)
Tricyclic carbazolequinones 10–12 showed weak CK2 inhib-
ition (% inhibition values 526%). Series of pyridocarbazolequi-
nones 13,14 and isoxazolocarbazolequinones 15,16 were not very
soluble at 10 mM in pure DMSO. Nevertheless, the saturated
solutions of derivatives 13, 15, and 16 were tested and showed
weak CK2 inhibitory activity (% inhibition values529%), having
in mind that these compounds could have stronger inhibitory
activities without the problem of solubility. In the series of
oxazinocarbazoles I–III, all compounds were soluble and the
assay was performed in triplicate. Two oxazino[5,6-c]carbazoles
Ib and Ig appeared to be potential CK2 inhibitors (% inhibition
values450%). They are structurally characterized by a short alkyl
chain in R1 (methyl for Ib and ethyl for Ig) and a common phenyl
group in R2. The IC50 values were calculated: 8.7 and 14.0 mM,
respectively.
Staphylococcus Enterococcus Pseudomonas Escherichia
faecalis
CIP 103214
aureus
Compound CIP 103429
aeruginosa coli
DSM 1117 DSM 1103
8
9
32
64
32
32
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
10
12
17
18
19
20
Ic
Ik
Il
16
16
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
4128
Ip
IIc
Further pharmacomodulation works were realized to optimize
these first results. In a recent SAR study34 on another tetracyclic
scaffold (indeno[1,2-b]indole), the best inhibitory activity against
CK2 was observed by introducing an isopropyl group on indolic
nitrogen. Then the oxazinocarbazole 2 exerted the best CK2
inhibition with an IC50 value of 1.40 mM (Table 2) and could be
used as lead compound for further structural exploration. In
contrast, the opening of the oxazine ring did not improve the
activity. In fact, aminomethylated carbazoles 3–7 showed a weak
level of inhibition (532%).
mitochondrial formazan converting enzymes only lose their
activity when the cell undergoes secondary necrosis with loss of
ATP and general proteolysis.
We conclude that the oxazinocarbazole derivatives were
indeed able to induce leukemia cell death at concentrations well
below 80 mM.
Oxazinocarbazoles as antibacterial agents
Oxazinocarbazoles as antileukemic agents
Most of the tested compounds did not display any significant
antibacterial activity at the tested concentrations (Table 3). Only
hydroxycarbazoles 8,9 and non-substituted carbazolequinone 10
were found to be somewhat effective, the latter being the most
efficient, but only on Gram-positive bacteria (i.e. S. aureus and
E. faecalis). These molecules had in common a low molecular
weight, as compared to the ineffective ones. Surprisingly,
compound 12, which only differs from compound 10 by the
addition of a hydroxyl substituent, lost all activity at concentra-
tions up to 128 mg/mL despite a rather low molecular weight.
The overall lack of efficiency on Gram-negative bacteria can be
hypothesized to be linked with an impermeability of the Gram-
negative cell wall to the tested compounds.
We tested the oxazinocarbazoles potency against IPC-81 leukemia
cells. The IPC-81 cell line is derived from the transplantable
BNML model of acute myelogenic leukemia (AML), known to be
a reliable predictor of the clinical efficiency of antileukemic
agents, like the first-line AML anthracycline drug daunorubicin
(DNR)35. The oxazinocarbazoles had IC50 values between 57 and
62 mM (Figure 8). All the tested compounds had a similarly steep
dose dependency, and were capable of inducing 100% IPC cell
death at concentrations above 80 mM (Figure 8). The cell death
morphology had apoptotic features, with nuclear condensation
and surface membrane blebs (not shown), which are typical for
such cells treated with various apoptosis inducers and anti-
leukemic drugs36. We also tested the cell’s mitochondrial
formazan conversion (WST-assay, see Methods section). This
assay agreed with the morphological assessment of cell death for
long incubation times. For short incubation periods, the micro-
scopic assay was superior, presumably because the already formed
Oxazinocarbazoles as antimalarial agents
Sixteen compounds were evaluated in vitro for their antimalarial
activity against P. falciparum 3D7 strain according with the IC50
values as shown in Table 4. As the strain used in this assay was

DOI: 10.3109/14756366.2014.899594
Biologically active carbazole derivatives 187
Table 4. Antiplasmodial activities (IC50 nM) of carbazole
derivatives 2, 8–10, 12, 17–20, Ib,c, Ig, Ik,l, Ip, and IIc.
and its congeners (e.g. oxazinocarbazoles) continue to be central
templates for drug design of new small molecules.
Plasmodium falciparum 3D7
IC50 (nM)
Acknowledgements
Compound
Dr. Z. Bouaziz and Pr. M. Le Borgne would like to gratefully thank
Christine Ranquet and Dr. L. Ettouati for their assistance in the stock
management of the small molecule library of EA 4446 B2C. We also
would like to thank ChemAxon for providing us license to their
cheminformatics software.
2
8
9
10
12
17
3380 108
0675 190
44500
44500
44500
44500
0551 181
44500
18
19
Declaration of interest
20
Ib
Ic
Ig
Ik
Il
Ip
44500
4244 653
44500
4131 482
44500
44500
0659 180
0430 390
018.9 0.53
The authors report no declarations of interest. We acknowledge financial
´
support to Zouhair Bouaziz and Marc Le Borgne by the ‘‘Bonus Qualite
Recherche’’ (BQR) of the University Claude Bernard Lyon 1, by the
´
‘‘Cluster 5 Chimie Durable et Chimie pour la Sante’’ of the Region
ˆ
´
ˆ
Rhone-Alpes and by the ‘‘ARC 1 Sante’’ of the Region Rhone-Alpes. The
‘‘Institut des Sciences Pharmaceutiques et Biologiques’’ (ISPB) is also
gratefully acknowledged for the funding of an uHPLC/DAD/MS system.
The present work was also supported by the ‘‘Partenariat Hubert
Curien’’ (PHC) (Campus France, Programme Aurora, Grant Agreement
No. 27460VC). Pr. Marc Le Borgne also thanks the ‘‘Institut franc¸ais
IIc
Chloroquine
˚
d’Oslo’’ for their support via the Asgard Programme 2010.
Dr. M. Baitiche is very grateful to the Ministry of Higher Education
and Research and to the University of Setif for sponsoring the trainings
well described, chloroquinosensible and chloroquine were used
for the validation of the assay. The susceptibility to available
antimalarial drugs for this strain following the used isotopic
method is close to the range 2–200 nM with end points
corresponding to dihydroartemisinin and quinine, respectively.
The susceptibility to doxycycline which is only used for malaria
prophylaxis is closed to 10 mM. Four compounds showed
interesting results with IC50 under 1 mM: hydroxycarbazole
derivative 8 (IC50 ¼ 675 nM), oxazinopyrimidocarboline deriva-
tive 18 (IC50 ¼ 551 nM) and two oxazinocarbazole derivatives Ip
(IC50 ¼ 659 nM) and IIc (IC50 ¼ 430 nM).
´
´
carried out at EA 4446 ‘‘Biomolecules Cancer et Chimioresistances’’.
We acknowledge financial support given to Samar Issa by the
´
‘‘Agence Universitaire de la Francophonie’’ (AUF) and the Region
ˆ
Rhone-Alpes (MIRA).
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