2-Substituted paullones: CDK1/cyclin B-inhibiting property and in vitro antiproliferative activity

Article abstract of DOI:10.1016/S0960-894X(00)00048-2

9-Trifluoromethyl-paullones with a carbon chain in the 2-position were synthesized by palladium-catalyzed coupling reactions of a 2-iodoprecursor with terminal alkenes or alkynes, respectively. The introduction of a 2-cyanoethyl substituent led to a significant enhancement of CDK1/cyclin B inhibiting property and in vitro antiproliferative activity. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00048-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 567±569
2-Substituted Paullones: CDK1/Cyclin B-Inhibiting Property and
In Vitro Antiproliferative Activity
Conrad Kunick, a,* Christiane Schultz, a Thomas Lemcke, a Daniel W. Zaharevitz, b
Rick Gussio, b Ravi K. Jalluri, b Edward A. Sausville, b Maryse Leost c
and Laurent Meijer c
aInstitut fur Pharmazie, Abteilung fur Pharmazeutische Chemie, Universitat Hamburg, Bundesstraûe 45, 20146 Hamburg,
Germany
È
È
È
bDevelopmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville,
MD 20852, USA
cCentre National de la Recherche Scienti®que, Station Biologique, BP 74, 29682 Rosco€, France
Received 8 October 1999; accepted 24 January 2000
AbstractÐ9-Tri¯uoromethyl-paullones with a carbon chain in the 2-position were synthesized by palladium-catalyzed coupling
reactions of a 2-iodoprecursor with terminal alkenes or alkynes, respectively. The introduction of a 2-cyanoethyl substituent led to a
signi®cant enhancement of CDK1/cyclin B inhibiting property and in vitro antiproliferative activity. # 2000 Elsevier Science Ltd.
All rights reserved.
The cyclin-dependent kinases (CDKs) are a family of
enzymes involved in cell cycle progression regulation.1
In a wide variety of human tumors and tumor cell
lines CDK-related mechanisms are deregulated.2 To
approach the possibility of treating neoplastic diseases
with synthetic CDK-inhibiting molecules, several struc-
tural classes of CDK inhibitors have been developed.3
The CDK inhibitor ¯avopiridol is currently undergoing
clinical trials as an anticancer drug.4 The paullones, a
series of indolo[3,2-d][1]benzazepines, have recently
been described as a novel class of small CDK-inhibiting
molecules related to the parent structure kenpaullone
1.5,6 Previous structure±activity relationship investiga-
tions revealed, that the 9-tri¯uoromethyl-paullone 2 is
equivalent to kenpaullone 1 with respect to CDK1
inhibition. However, both 1 and 2 exhibited only poor
antiproliferative activity.7 A computer model was con-
structed, in which kenpaullone 1 is bound to the ATP
binding site of CDK2.6 Based on this model, it was
postulated that substituents in the 2-position of the
paullone sca€old should be accommodated in the access
channel to the ATP binding cleft. Polar terminal groups
at the introduced carbon chains were envisaged to form
favorable interactions with solvent molecules or amino
acid side chains in the vicinity of the ATP binding site
entrance. To explore this hypothesis, 9-tri¯uoromethyl-
paullones with appropriate 2-substituents were prepared
and tested for CDK1/cyclin B inhibition and in vitro
antitumor activity.
Chemistry
The 2-iodo-9-tri¯uoromethyl-paullone 8 was prepared
according to a method for the synthesis of similar 7,12-
dihydro-indolo[3,2-d][1]benzazepin-6(5H)-ones.8,9 Thus,
5-iodoanthranilic acid 3 was converted to the ethyl ester
4, which was subsequently reacted with ethyl succinyl
chloride to furnish the amide 5. A Dieckmann cycliza-
tion was then accomplished by means of potassium
*Corresponding author. Fax: +49-40-42838-6573; e-mail: kunick@
chemie.uni-hamburg.de
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00048-2

568
C. Kunick et al. / Bioorg. Med. Chem. Lett. 10 (2000) 567±569
hydride, and the resulting ester 6 was dealkoxy-
carbonylated by heating in wet DMSO. The 7-iodo-1H-
[1]benzazepine-2,5-(3H,4H)-dione 7 yielded the paul-
lone 8 by a Fischer ring closure with 4-tri¯uoromethyl-
phenylhydrazine (Scheme 1).
National Cancer Institute (NCI) on 60 human tumor
cell lines.13,14 The results of this screening are processed
to give an averaged parameter over all cell lines for each
test compound, designated as meangraph midpoint
(MGM).15 In Table 1, the IC50 values for CDK1/cyclin
B and the MGMs for the paullones 8±11 are reported
and compared to kenpaullone 1 and its analogue 2.
Results on the colon cancer cell line HCT-116 are
included, because this line proved to be especially sensi-
tive to the paullones 1 and 2.7
Subsequent Heck reaction of 8 with terminal alkenes
under standard conditions10 a€orded the E-con-
®gurated 2-substituted paullones 9a±c. The saturated
analogue 11 was prepared upon re¯uxing the acrylo-
nitrile derivative 9c with magnesium turnings in metha-
nol.11 The reaction of terminal alkynes with 8 in the
presence of cuprous iodide and a palladium catalyst in
triethylamine furnished the 2-alkynyl-paullones 10a and
10b (Scheme 2).12
All newly prepared paullones turned out to be con-
siderably active as CDK1/cyclin B inhibitors, support-
ing the hypothesis that larger substituents in the 2-
position of paullones are tolerated. However, there were
noteworthy di€erences in potency depending on the
nature of the substituent. A loss of one order of magni-
tude in potency was detected for the structures 9a
and 10b with respect to kenpaullone 1. For 10b the
decreased potency might be accounted for by the bulky
cyclohexyl group, which is kept in position by the rigid
carbon chain at the paullone sca€old. Accordingly, the
analogue 10a lacking the cyclohexane ring turned out to
be equipotent to 1 and 2. To explain the low potency of
Biological Evaluation and Discussion
The paullones 8±11 were tested in a CDKl/cyclin B
kinase inhibition assay described previously, using
CDKl/cyclin B from star®sh oocytes and histone H1 as
substrate.7 The in vitro antitumor activity was deter-
mined in the anticancer drug discovery screen of the
Scheme 1. Synthesis of 2-iodo-9-tri¯uoromethyl-paullone 8. Reagents and conditions: (i) EtOH, gaseous HCl, Á, 7 h; (ii) ClCO±(CH2)2±CO2C2H5,
toluene, pyridine, Á, 2 h; (iii) KH, toluene, DMF, N2, 0 ꢀC!80 ꢀC, 3 h; (iv) DMSO, H2O, 150 ꢀC, N2, 3 h; (v) 4-tri¯uoromethyl-phenyl-hydrazine,
AcOH, 70 ꢀC, 1 h; (vi) H2SO4, AcOH, 70 ꢀC, 1 h.
Scheme 2. Synthesis of 2-substituted paullones from 8. Reagents and conditions: (i) R-CHˆCH2, Pd(OAc)2, P(C6H5)3, TEA, DMF, N2, Á, 4±14 h;
(ii) R-CꢁCH, PdCl2[P(C6H5)3]2, CuI, TEA, N2, 50 ꢀC, 1±5 h; (iii) Mg, MeOH, Á, 1 h.

C. Kunick et al. / Bioorg. Med. Chem. Lett. 10 (2000) 567±569
Table 1. CDK1/cyclin B inhibition and in vitro antitumor activity
569
a
log10 GI50
Compound
mp (ꢀC)
IC50 CDK1/cyclin B (mM)b
HCT-116 (M)c,d
MGM (M)d,e
1
2
8
9a
9b
9c
10a
10b
11
>330 (1,4-dioxane)
>330 (EtOH)
>330 (EtOH)
>330 (EtOH/toluene)
>330 (EtOH)
>330 (EtOH)
>330 (EtOH)h
>330 (EtOH)i
286 (dec.; EtOH)
0.4
0.4
0.7
4.3
0.32
0.27
0.3
3.2
0.047
5.7/ 5.7
5.4
> 4.0
NAf
5.9/ 5.8g
> 4.0
5.5/ 5.6
5.6/ 5.7
6.1/ 5.7
4.4/ 4.3
4.1
4.07
4.02
5.6/ 5.3g
4.08
4.9/ 4.8
5.7/ 5.6
5.7/ 5.6
4
aGI50=concentration for 50% growth inhibition. The highest concentration used in the in vitro antitumor tests was 10 M.
bTests were carried out in triplicate.
cColon cancer cell line.
dResults of two test runs are separated by a slash.
eMGM=meangraph midpoint.
fNA=not available.
gOne set of test results omitted due to high deviation.
hDecomposition starting at 300 ꢀC.
iDecomposition starting at 275 ꢀC.
9a with respect to 9b, a preliminary computer model
was constructed for the CDK-binding of both com-
pounds showing that the vinyl ketone's end methyl
group of 9b was within the favorable binding region
of the pocket, whereas the vinyl ester's end methyl
group of 9a protrudes into the solvent front to about 1 A
outside the binding site. Thus, the larger entropic
penalty conferred by the slightly bulkier ester may
account for the diminished potency of 9a. In a similar
model, the cyano nitrogen's lone pair of 9c was favor-
ably extended into the solvent. The remarkable increase
in CDK1/cyclin B inhibitory potency of the saturated
analogue 11 with regard to 9c might either be the result
of the higher ¯exibility of the side chain or of the lost
conjugation between the cyano group and the paullone
ring system. As far as 2-substituted paullones of the
present study are concerned, the in vitro antitumor
activity is not paralleling the CDK1 inhibitory property.
Hence, the acrylonitrile-substituted derivative 9c is
nearly inactive as antiproliferative agent, although it
exhibits CDK1 inhibition in submicromolar concentra-
tions. On the other hand, 10b shows lower CDK1 inhi-
bition compared to 9c, but remarkable antitumor
activity. The 2-cyanoethyl derivative 11 satis®es the
main rationale of this study, being a potent CDKl inhi-
bitor as well as an interesting in vitro antitumor agent.
In conclusion, the 2-substitution of the paullones
appears to be a promising strategy for the development
of antiproliferative agents. However, in the course of
future studies it should be taken into consideration that
other targets besides the CDKs might account for the
antiproliferative activity of the paullones.
pour la Recherche sur le Cancer'' (ARC 9314) (to L.M.)
and the ``Conseil Regional de Bretagne'' (to L.M.) and
by a C.N.R.S./NCI collaboration contract. We thank
the ®shermen of the ``Station Biologique de Rosco€''
for collecting the star®sh. Computer time and software
was provided, in part, by the Advanced Biomedical
Computing Center, Frederick, MD.
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Acknowledgements
The generous support by Professor D. Ge€ken and the
University of Hamburg is gratefully acknowledged. This
research was supported by grants from the ``Association