Pyrrolo[2,3-a]carbazoles as potential cyclin dependent kinase 1 (CDK1) inhibitors. Synthesis, biological evaluation, and binding mode through docking simulations

Article abstract of DOI:10.1021/jm0700666

Pyrrolo[2,3-a]carbazole derivatives were synthesized, and their effects on CDK1/cyclinB activity were evaluated. The most potent and efficacious inhibitor was found to be ethyl 9-chloro-1H-pyrrolo[2,3-α]carbazole-2-carboxylate (1e), exhibiting an IC₅₀ in the low micromolar range and leading to 90% at higher concentrations. Using a computational model for CDK1 - 1e, binding we have observed that 1e exhibited two likely binding modes in the ATP-binding cleft that involve interactions with Lys130, Thr14, and Asp146 of the enzyme.

Full text of DOI:10.1021/jm0700666

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J. Med. Chem. 2008, 51, 1048–1052
Pyrrolo[2,3-a]carbazoles as Potential Cyclin Dependent Kinase 1 (CDK1) Inhibitors. Synthesis,
Biological Evaluation, and Binding Mode through Docking Simulations
Manolis A. Fousteris,† Athanasios Papakyriakou,‡ Anna Koutsourea,† Maria Manioudaki,§ Evgenia Lampropoulou,§
Evangelia Papadimitriou,§ Georgios A. Spyroulias,‡ and Sotiris S. Nikolaropoulos*,†
Laboratory of Medicinal Chemistry, Laboratory of Pharmacognosy and Chemistry of Natural Products, Laboratory of Molecular
Pharmacology, Department of Pharmacy, School of Health Sciences, UniVersity of Patras, GR-26500 Rion, Patras, Greece
ReceiVed January 17, 2007
Pyrrolo[2,3-a]carbazole derivatives were synthesized, and their effects on CDK1/cyclinB activity were
evaluated. The most potent and efficacious inhibitor was found to be ethyl 9-chloro-1H-pyrrolo[2,3-
R]carbazole-2-carboxylate (1e), exhibiting an IC50 in the low micromolar range and leading to 90% at higher
concentrations. Using a computational model for CDK1-1e, binding we have observed that 1e exhibited
two likely binding modes in the ATP-binding cleft that involve interactions with Lys130, Thr14, and Asp146
of the enzyme.
Introduction
derived from these complexes has elucidated key interactions
needed for effective inhibitor binding; these data have greatly
contributed to the design and optimization of new lead
compounds. Moreover, sequence alignment and homology
modeling have revealed a high degree of similarity among
CDK2 and CDK1.
Cyclin dependent kinases (CDKs), a family of serine/
threonine kinases, play a pivotal role in the regulation of cell
cycle progression. In addition, CDKs are also implicated in
apoptosis, neuronal cell physiology, differentiation, and tran-
scription.1 Enzymatic activity of CDKs is tightly regulated by
several means including binding to cyclins, phosporylation by
CDK activating kinases (CAKs), dephosphorylation by phos-
phatases, and association with negative regulatory proteins
(CDK inhibitors, CKIs).2 Among the 11 standard CDKs that
have been identified in the human genome,3 CDKs 1-4 and 6
have been proposed to control the transition from one stage of
the cell cycle to the next after forming complexes with the
appropriate cyclin subunit. Deregulation of CDKs and their
modulators occurs in many human tumors4 and in a number of
proliferative and neurodegenerative5 disorders. Thus, CDKs
represent attractive targets for therapeutic intervention. Cur-
rently, much effort has focused on the discovery of new
chemical inhibitors of CDKs that could act as anticancer agents.
Up to date, several scaffolds have been recognized as efficient
inhibitors of these enzymes,6,7 with several of them being in
the late phases of clinical trials.8,9 Purines, pyrimidines, fla-
vonoids, indolocarbazole analogues, and other heterocyclic
compounds (butyrolactone, fascaplysin, paullones) comprise
typical representatives with well established CDK inhibitory
activity. Particularly, most of the already known CDK inhibitors
(natural products or/and synthetic molecules) are flat, small
heterocycles that act by competing with ATP for the kinase
ATP-binding site. Although some inhibit CDK activity in
nanomolar concentrations, optimizing potency and selectivity
against individual CDK family members remains a challenge.
Initial structure-based drug discovery efforts focused on CDK2
inhibition, as it was the first member of CDK family for which
the crystal structure was resolved.10 Several small molecules
have been cocrystallized with CDK2,11–15 and information
On the basis of the above-mentioned data and considering
that CDK1 is sufficient to drive the mammalian cell cycle,16
we aimed at synthesizing new compounds with potential CDK1
inhibitory activity. We considered pyrrolo[2,3-a]carbazole core
1 as a promising scaffold, as it bears structural features that
could potentially contribute to the development of interactions
with the ATP-binding site of CDKs. Herein, we are reporting
a slightly modified synthesis method for pyrrolo[2,3-a]carbazole
analogues17 along with preliminary results concerning their
ability to inhibit CDK1 in vitro. Moreover, using a docking
simulation approach, we have tried to address the structural basis
of CDK1-inhibitor interactions.
Results and Discussion
Chemistry. The general methodology for the synthesis of
pyrrolo[2,3-a]carbazole analogues is outlined in Scheme 1. The
key step for the construction of the functionalized tetracyclic
pyrrolocarbazole core involves Fischer indolization of tetrahy-
droindole ketone 3 with various arylhydrazine derivatives.
Previously reported17 synthetic approach to pyrrolo[2,3-
a]carbazole core was based on the employment of the tetrahy-
droindole ketone 218 as building block in Fischer indolization
with various arylhydrazines. However, the fragility of ketone
2’s corresponding arylhydrazones prompted us to explore the
utilization of a protective group for the pyrrole nitrogen atom.
The inserted group was expected to contribute to the stabilization
of the pyrrole ring and hence to the increased stability of
arylhydrazones under the subsequent Fischer indolization condi-
tions. For this approach, the application of the p-methoxybenzyl
* To whom correspondence should be addressed. Phone: +302610969326.
Fax: +302610992776. E-mail: snikolar@upatras.gr.
†
Laboratory of Medicinal Chemistry.
Laboratory of Pharmacognosy and Chemistry of Natural Products.
Laboratory of Molecular Pharmacology.
‡
§
a Abbreviations: PMB, p-methoxybenzyl group; PPSE, polyphosphoric
acid trimethylsilyl ester; TFA, trifluoroacetic acid; UCN01, 7-hydroxy-
staurosporin.
10.1021/jm0700666 CCC: $40.75
2008 American Chemical Society
Published on Web 01/31/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1049
Scheme 1a
a Conditions. (i) t-BuOK, 18-crown-6, PMB-Br or Bn-Br, Et2O, room temp, 2.5 h; (ii) ArNHNH2 ·HCl, AcONa, EtOH/TFA, reflux, 2–4 h; (iii) PPSE,
CH3NO2, reflux, 4–44 h. (iv) R1 ) PMB: anisole, H2SO4, TFA, 0 °C, 0.5 h and then room temp, 2–3.5 h. (v) R1 ) Bn: AlCl3, C6H6, 0 °C, 15 min and then
50 °C, 0.5 h.
(PMBa) moiety as a protecting group for the pyrrole nitrogen
atom was initially investigated, considering that the latter has
beenusedsuccessfullyinthechemistryofnitrogenheterocycles.19–21
Formation of the PMBa ketone 3 was achieved according to
the method of Guida and Mathre22 with minor modifications.
Specifically, treatment of 2 with freshly prepared p-methoxy-
benzyl bromide (PMB-Br)23 in the presence of potassium tert-
butoxide (t-BuOK) and 18-crown-6 in diethyl ether under phase
transfer conditions afforded the N-PMB ketone 3 in good yield
(75%). Condensation of 3 with various substituted arylhydra-
zines in the presence of anhydrous sodium acetate and trifruo-
roacetic acid in ethanol afforded the intermediate arylhydrazones
4a-f. Subsequently, following an one-pot procedure, the crude
mixture of 4a-f was employed in Fischer cyclization conditions
using freshly prepared polyphosphoric acid trimethylsilyl ester
(PPSE) as an aprotic catalyst and nitromethane as a solvent.17,24
Only the N(1)-PMB pyrrolo[2,3-a]carbazole derivatives 5a-f
were obtained in all cases, in contrast to our previously reported
method17 where the Fischer indolization of the ketone 2’s
corresponding arylhydrazones gave the desired fully aromatized
products accompanied by the corresponding 4,5-dihydro ones.
Finally, cleavage of the protective group by treatment with TFA
in the presence of anisole and concentrated sulfuric acid19
afforded the final products 1a-f in very good yields (83–96%).
The benzyl (Bn) group was alternatively investigated as a
possible protective group for ketone 2, but it proved to be
inferior to the PMB group because the debenzylation proce-
dure25 of the N(1)-benzylpyrrolo[2,3-a]carbazole derivatives 8a
and 8e led to a complex mixture of the unprotected final products
and polyalkylated byproduct. Isolation of the desired derivatives
1a and 1e proved to be insufficient with the conventional
Figure 1. (A) Effect of the tested compounds at 100 µM on the activity
methods, and it was accomplished only using HPLC chroma-
tography. Thus, the PMB group facilitated the preparation of
the functionalized pyrrolocarbazole core in an efficient and
convenient manner without affecting functional groups of target
molecules or rendering their isolation unattainable. Moreover,
it contributed to the complete aromatization of the Fischer
indolization products and to the avoidance of the subsequent
aromatization process involved in the previously reported
method.17
Biological Activity. Compounds 1a-f were tested for their
inhibitory activity in vitro against the CDK1/cyclin B complex
using the MESACUP cdc2 kinase assay kit (see Supporting
Information). Compounds 1a-c, 1d1, 1d2, and 1f were inactive
of CDK1. (B) Concentration-dependent inhibition of CDK1 by 1e.
Results are expressed as the mean ( SEM of the % CDK1 activity.
Asterisks in all cases denote a statistically significant difference from
the untreated samples (control): (/) P < 0.05; (///) P < 0.001.
against CDK1/cyclin B (Figure 1A). Interestingly, 1e showed
a concentration-dependent, statistically significant inhibitory
activity against CDK1/cyclin B (Figure 1). As estimated from
Figure 1B, the concentration of 1e that caused CDK1 inhibition
by 50% was 15 µM, while the reference compound olomoucine
exhibits an inhibitory activity against the same complex with
IC50 ) 7 µM.26
Moreover, we tested the 1e against the CDK1/cyclin B1
complex, using an additonal in vitro kinase reaction approach

1050 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Brief Articles
site. With respect to the conformer with the highest docked
energy (1e-I), the compound’s second placement (1e-II) (Figure
2B,C) resulted in the overlap of three out of four rings in a
way that (i) rings B are well fitted and (ii) ring A of 1e-I is
well fitted to ring C of 1e-II and vice versa (ring C of 1e-I is
perfectly fit ted to ring A of 1e-II). The two orientations differ
in terms of docked energy by less than 0.8 kcal/mol. Among
the seven compounds studied here, only 1e exhibits remarkably
high inhibition properties, up to 90% inhibition of CDK1
(Figures 1 and 2B,C).
The most important CDK1-substrate simulated interactions
involve residues Ile10, Glu12, Gly13, Thr14, Tyr15, Val18,
Ala31, Lys33, Val64, Phe80, Glu81, Phe82, Leu83, Asp86,
Asp128, Lys130, Gln132, Asn133, Ala145, Asp146, and Leu149
(Figure 2B,C). In all cases, Val18, Lys33, Lys130, Asn133,
Ala145, and Asp146 are predicted to exhibit major contacts with
the pyrrolo[2,3-a]carbazole core inside the ATP-binding cleft.
These residues define the following substrate binding subsites:
(i) a hydrophilic region consisting of residues Glu12-Tyr15, (ii)
the polar area comprising Asp128, Lys130, Gln132, and Asn133,
(iii) the Asp146 docking site favorable for H-bond formation,
and (iv) the Phe80 to Leu83 fragment.
Figure 2. (A) Ribbon representation of CDK1 homology model
generated with MOLMOL.32 Docked 1 conformers of lowest energy
are illustrated to be in the ATP-binding cleft. Side chains of CDK1
residues involved in CDK1-1 interaction (within 4.0 Å) are also shown
(residues Glu12, Gly13, Thr14, Tyr15, Val18, Ala31, Lys33, Val64,
Phe80, Asp128, Lys130, Gln132, Asn133, Asp146, (B, C) Views
differing by 90° of the lowest energy conformation for 1d2 in light
blue and for 1e in blue, anchored to the CDK1 ATP-binding cleft.
Residues that surround the ligands and atoms of the pyrrolo[2,3-
R]carbazole derivatives involved in the CDK1-ligand interactions (B)
are denoted. Residues that are found within 4 Å from the ligands are
shown as yellow sticks (C).
According to the analysis of the CDK2-ATP crystal struc-
ture,30 the corresponding CDK1 residues that might be important
for ATP-binding are Asp86 and Gln132 (Gln131 in CDK2) and
Lys33 and Asp146 (Asp145 in CDK2). From those, Lys33,
Lys130, and Asp146 are predicted to possess a key role in
positioning and binding of 1e through hydrogen bonds (Figure
2B,C). It is noteworthy that N1 and N10 of the pyrrolo[2,3-
a]carbazole scaffold are predicted to be in a favorable position
to form hydrogen bonds with side chain carboxylic oxygen
atoms of Asp146. In CDK1-1e-I simulated complex Asp146
Oδ2 is in H-bond distance with N10 and N1 atoms (2.6 and
2.9 Å). In contrast, in CDK1-1e-II complex Asp146 both
carboxylic oxygens are predicted to be close to both pyrrole
nitrogens of the ligand; Oδ1 atom is at 3.2 and 4.2 Å to N1
and N10, respectively, and Oδ2 at 2.5 and 2.6 Å to N10 and
N1, respectively (Figure 2B,C). An examination of all available
CDK2-inhibitor crystal structures7 has revealed that such an
interaction between both carboxylic oxygens of Asp145 (Asp146
in CDK1) and inhibitor is not present. These data underline the
role of Asp145 in CDK1-inhibitor complexes, suggesting a
tight interaction of 1e active compound presented herein with
ATP-binding cleft in CDK1. Asp146 (Asp145 in CDK2), which
has been reported to be coordinated to Mg2+ and to make
contact with the ATP triphosphate group through the metal ion
in the CDK2-ATP complex, is far from staurosporine in
CDK2-staurosporine complex (∼6.0 Å to the closest stauro-
sporine atom). The same residue in CDK2-UCN01 complex
is H-bonded through its backbone nitrogen to a water molecule,
which is found to be close to the 7-hydroxyl group of UCN01.
(see Supporting Information). This compound inhibited CDK1
activity by 50% when used at 30 µM. 1e also proved to be
inactive against CDK1/cyclin A and CKD1/cyclin E complexes.
To determine whether 1e also affects the activity of receptor
tyrosine kinases, we tested it on EGFR, IGFR, FGFR, KDR,
Flt-4, and Tie2 (data not shown). In all cases 1e proved to be
inactive up to 50 µM using a previously described methodol-
ogy.27
Molecular Modeling. Interaction of pyrrolo[2,3-a]carbazoles
derivatives and CDK1 was studied through molecular modeling
and docking simulations using a CDK2-based homology mode
of CDK1.28 CDK1 (297 residues) exhibits 63.6% sequence
identity with CDK2 (298 residues; see Supporting Information).
The structure of CDK2 in complex with ATP (PDB code
1B38)29 was chosen with the criterion that it is the best
representative model to address the ATP binding site. The ribbon
representation of the energy minimized CDK1 model is shown
in Figure 2A.
Focusing on the most potent derivative 1e, it is noted that in
simulated 1e-I placement the ligand exhibits some H-bond
interactions with (i) side chain amine protons of Lys33, (ii) the
Thr14 amide HN and the O1 (CdO group), (iii) side chain
amine protons of Lys130 and the O2 oxygen of all compounds,
while it accommodates the carboxyl ester -CH3 toward the
aromatic ring of Tyr15 and between the backbone of Asp128
and the side chains of Asp146. The CdO moiety is close to
On the basis of the homology model of CDK1, efforts were
focused on the generation of the protein–substrate complexes
through a docking simulation approach. All seven compounds
(see Supporting Information for experimental details) exhibit a
remarkable preference for binding to the ATP-binding cleft. The
compounds used are classified according to the substitution at
the D ring of the pyrrolo[2,3-a]carbazole core and the nature
of the substituent. All derivatives exhibit docking energies from
-11.0 to -12.8 kcal/mol, with 1a exhibiting the lowest docking
energy. Additionally, derivative 1e, which proved to be the most
potent inhibitor of CDK1 as described above, exhibited the
highest docking energy. However, docking simulations of 1e
suggest two likely placements into the CDK1 ligand binding
+
Lys130 side chain -NH3 group with distance and geometry
favorable for H-bonding. In such a conformation the chlorine
atom of 1e-I orients toward the Phe80 ring (in 3.4 Å distance).
In CDK1-1e-II simulated complex, the ligand orients its
carboxyl ester chain perpendicular to the aromatic moiety close

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1051
dried over anhydrous sodium sulfate. The solvent was evaporated.
The crude residue was purified by column chromatography using
toluene/ethyl acetate gradient as eluent (from 100/0 to 90/10) to
afford the title compounds 5a-f and 8a,e.
to Val18, Lys33, and Phe80, while the chlorine atom is found
+
close (∼3.6 Å) to the positively charged -NH3 group of
Lys130.
Ethyl 9-Chloro-1-(4-methoxybenzyl)-1H,10H-pyrrolo[2,3-a]-
carbazole-2-carboxylate (5e). Reaction time: 15 h. 21% yield. IR
Conclusions
(film): 3391, 1702, 1244, 813, 739 cm-1 1H NMR (CDCl3): δ
.
While aiming to generate agents with inhibitory CDK1
activity, we designed and synthesized, following a modified
route, a novel series of compounds bearing the pyrrolo[2,3-
a]carbazole core. Our initial design was confirmed by biological
results where one of the studied derivatives (1e) exhibited
inhibitory potency against CDK1/cycline B, causing 50%
inhibition when used at 30 µM. At concentrations up to 100
µM the rest of the compounds proved to be inactive, while 1e
at this high concentration caused 90% inhibition. In subsequent
experiments, 1e proved to be inactive against a panel of kinases
(CDK1/cyclin A, CDK1/cyclin E, EGFR, IGFR, FGFR, KDR,
Flt-4, and Tie2) when used at 50 µM, demonstrating selectivity
toward the CDK1/cycline B complex.
By use of a homology model of CDK1, CDK1-pyrrolo[2,3-
a]carbazole complexes were modeled. The structural analysis
of the simulated complexes attempts to illustrate the physico-
chemical and structure determinants for 1e’s high inhibitory
potential toward CDK1. Additionally, this study provides a
different interaction platform between CDK1 and a potential
inhibitor bearing the pyrrolo[2,3-a]carbazole scaffold. Since no
X-ray structure of CDK1 is available, all data for structure-
based inhibitor design are based on CDK2-substrate complexes
and on simulated complex of CDK1 homology models with
heterocycle ligands.7,29,30 According to these studies, substrate
binding into ATP binding pocket of CDK2 implicates Lys33,
Glu81, Leu83, Asp86, and Gln131 and according to CDK1-
paullone complexes, Glu8, Lys20, Leu83, Asp86, Lys89, and
Gln300.31 The pyrrolo[2,3-R]carbazole derivative 1e docking
studies suggest that this compound does not preferentially fit
into the Glu81-Leu83 cleft but exploits remarkably the potential
of Lys130, Thr14, and Asp146 side chains in positioning and
binding. Structural modifications of the basic pyrrolo[2,3-
R]carbazole core are under investigation aiming at the deter-
mination of structure–activity relationships and optimization of
the inhibitory potency or/and selectivity, and they will be
reported in due course.
8.19 (br s, 1H), 7.84 (d, 1H, J ) 7.8 Hz), 7.72 (d, 1H, J ) 8.5 Hz),
7.48–7.45 (m, 2H), 7.24–7.18 (m, 3H), 7.07 (t, 1H, J ) 7.8 Hz),
6.85 (d, 2H, J ) 8.6 Hz), 6.09 (s, 2H), 4.33 (q, 2H, J ) 7.1 Hz),
3.69 (s, 3H), 1.35 (t, 3H, J ) 7.1 Hz). 13C NMR (CDCl3): δ 162.0,
159.4, 136.0, 130.2, 127.4, 127.3, 127.1, 125.7, 125.4, 125.1, 123.8,
120.9, 120.6, 118.1, 116.5, 115.3, 115.2, 114.5, 112.6, 60.7, 55.3,
49.3, 14.4. LC-MS (ESI+) m/z 433.10 [M + H+]. Anal.
(C25H21ClN2O3) C, H, N.
General Procedure for the Preparation of the
Pyrrolo[2,3-a]carbazole Derivatives 1a-f. To a stirred mixture
of the protected 5a-f (0.05 mmol) in trifluoroactic acid (0.6 mL)
at 0 °C under argon atmosphere, anisole (0.136 mmol, 148 µL)
and concentrated sulfuric acid 7.5 µl) were added. The mixture
was stirred 0.5 h at 0 °C and then for 2–3.5 h at room temperature.
The solution was then added dropwise to an ice-cooled solution of
saturated aqueous sodium bicarbonate (3 mL), and the mixture was
extracted with ethyl acetate three times. The combined organic
extracts were washed with brine, dried over anhydrous sodium
sulfate, and concentrated in vacuo. The crude product was purified
by column chromatography using toluene/ethyl acetate gradient as
eluent (from 100/0 to 80/20) to give the unprotected products 1a-f.
The analytical and spectroscopic data of the title compounds were
identical to those previously reported.17
Ethyl 9-Chloro-1H,10H-pyrrolo[2,3-a]carbazole-2-carboxy-
late (1e). White solid, 95% yield. Mp 285–286 °C (EtOAc).
1
IR(KBr): 3427, 3288, 1682, 1206, 748 cm-1. H NMR (DMSO-
d6): δ 11.67 (s, 1H, NH), 11.26 (s, 1H, NH), 8.10 (d, 1H, J ) 7.8
Hz), 7.85 (d, 1H, J ) 8.5 Hz), 7.52–7.43 (m, 2H), 7.34 (s, 1H),
7.22 (t, 1H, J ) 7.8 Hz), 4.40 (q, 2H, J ) 6.8 Hz), 1.39 (t, 3H, J
) 7.1 Hz). 13C NMR (DMSO-d6): δ 161.0, 134.8, 125.8, 125.7,
125.4, 123.8, 123.3, 120.1, 118.5, 118.3, 115.3, 114.2, 114.0, 109.3,
109.2, 60.5, 14.2. LC-MS (ESI+) m/z 312.88 [M + H+]. Anal.
(C17H13ClN2O2) C, H, N.
Acknowledgment. We thank the European Social Fund
(ESF), Operational Program for Educational and Vocational
Training II (EPEAEK II), and particularly the Program PYTHAG-
ORAS II, for funding this work. The authors are deeply indebted
to Dr. Jonathan Pines and Dr. Mark Jackman of Wellcome/
Cancer Research U.K. Institute, Cambridge, U.K., for perform-
ing the phosphoimager experiments and to Prof. Dr. Athanassios
Giannis of Institute of Organic Chemistry, Leipzig University,
for performing the in vitro experiments for the rest of the
kinases.
Experimental Section
The target compounds 1a-f have been synthesized previously17
using a different synthetic approach.
General Procedure for the Preparation of Arylhydrazones
4a-f and 7a,e. To a solution of ketone (3 or 6) (0.61 mmol) in
absolute ethanol (4.3 mL) was added trifluoroacetic acid (2.38
mmol, 0.18 mL), and the mixture was stirred at room temperature
under an argon atmosphere. A suspension of the appropriate
arylhydrazine (1 mmol) and sodium acetate anhydrous (1.22 mmol,
100 mg) in absolute ethanol (4.3 mL) was added dropwise over 15
min. The mixture was stirred at 80 °C for 2–4 h under argon
atmosphere. After the completion of the reaction (TLC monitoring),
solvents were removed under reduced pressure, dichloromethane
(5 mL) was added, and evaporation was repeated. The crude mixture
was dried in vacuo.
Supporting Information Available: Synthetic procedures,
spectroscopic data for new compounds, biological assays details,
and computational methods. This material is available free of charge
via the Internet at http://pubs.acs.org.
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