Novel CDK inhibition profiles of structurally varied 1-aza-9-oxafluorenes

Article abstract of DOI:10.1016/j.bmcl.2004.10.091

A series of 1-aza-9-oxafluorenes with functionally varied 3-substituents have been prepared from N-phenoxycarbonyl-4-phenyl-1,4-dihydropyridines and p-benzoquinone and biologically evaluated as inhibitors of various cyclin-dependant kinases. The absence o

Full text of DOI:10.1016/j.bmcl.2004.10.091

Bioorganic & Medicinal Chemistry Letters 15 (2005) 823–825
Novel CDK inhibition profiles of structurally varied
1-aza-9-oxafluorenes
Burkhardt Voigt,a Laurent Meijer,b Olivier Lozach,b Christoph Scha¨chtele,c
Frank Totzkec and Andreas Hilgerotha,*
aInstitute of Pharmaceutical Chemistry, Department of Pharmacy, Martin-Luther-University Halle-Wittenberg,
Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany
bCNRS, Cell Cycle Group, Station Biologique, BP 74, Roscoff 29682 Cedex, France
cProQinase GmbH, Breisacher Strasse 117, 79106 Freiburg, Germany
Received 15 March 2004; revised 22 October 2004; accepted 28 October 2004
Available online 2 December 2004
Abstract—A series of 1-aza-9-oxafluorenes with functionally varied 3-substituents have been prepared from N-phenoxycarbonyl-4-
phenyl-1,4-dihydropyridines and p-benzoquinone and biologically evaluated as inhibitors of various cyclin-dependant kinases. The
absence of a 3-hydrogen bond acceptor function leads to a complete loss of inhibitory activity. Differing hydrogen bond acceptor
functions surprisingly cause significant shifts in the selectivity of inhibition profiles.
Ó 2004 Elsevier Ltd. All rights reserved.
Cyclin-dependant kinases (CDKs) play a key role in the
cycle of dividing cells.1 Associated with differing cyclins
and by specific natural protein inhibitors they control
the progression of cell growth and division.2–4 Occurring
mutations lead to uncontrolled growth as well as cell
degeneration process with resulting diseases like cancer
or AlzheimerÕs disease (AD), for which each specific kin-
ases have been characterized as responsible agents.5,6
7 inhibitory activity, or substituted purine compounds
2, which also inhibit CDK5/p251 (Fig. 1).
OH
O
1
R
R
HN
N
Therefore CDKs like 1, 2 and 4 have been target en-
zymes for the development of small-sized inhibitors with
antiproliferative properties in cancer progression.5
CDK5/p25 plays a central role in ongoing AlzheimerÕs
disease by phosphorylation of tau-protein leading to
the formation of neurofibrillary tangles and neuronal
decline. Inhibitors of this kinase have been investigated
as potential AD therapeutics.6
O
(X)
n
HO
N
N
3
R
OH
N
HO
N
H
N
2
R
X = O, S
n = 0, 1
CH3
1
2
H
N
O
X4
Y1
The main problem in the development of such small-
sized inhibitors is the selectivity of the inhibition.1,5
Among those various reported classes of CDK inhibi-
tors, some show selectivity profiles for CDKs like substi-
tuted Flavopiridol-analogues 1, with some preference
for both CDK1 and 2 inhibition beside CDK4, 6 and
HO
O
R
H
N
O
N
Br
4a, R = CH3
elbfluorene
3a, X = C, Y = N
3b, X = N, Y = C
*
Corresponding author. Tel.: +49 345 55 25168; fax: +49 345 55
27026; e-mail: hilgeroth@pharmazie.uni-halle.de
Figure 1.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.091

824
B. Voigt et al. / Bioorg. Med. Chem. Lett. 15 (2005) 823–825
Reduced CDK-inhibitory activities were recently re-
ported for 1-azakenpaullone 3a compared to 4-azaken-
paullone 3b with similar activities against CDK1 and
5/p25 as well as glycogen synthase kinase (GSK)-3b,7 a
kinase involved in various cell cycle regulation processes
and with dysregulation in AD progression. The resulting
observed selectivity of 3a as GSK-3b inhibitor is caused
by the modified binding properties of the 1-azacom-
pound compared to the 4-azacompound because the
nitrogen atom responsible for potential hydrogen bond
acceptor function to the enzyme backbone is situated
at the opposite molecular site.
+
R
R
R
(i)
-
Cl
N
N
O
O
5a-e
Comp.
a
CH3
OCH3
OC2H5
Cl
R
b
c
d
e
We recently presented 1-aza-9-oxafluorenes 4 as a novel
class of small-sized cytostatics.8 A Compare-analysis of
one 4-phenyl derivative with NCI-database compounds
gave the first hints to possible kinase inhibitory proper-
ties as mode of cytostatic action of the nonplanar mole-
cules. Those 3-acyl substituted candidates turned out
primarily as selective CDK1 inhibitors compared to
other CDKs like CDK2 and CDK4 or CDK5.9
(ii)
N
O
O
Br
6a-e
The 6-hydroxy function was found essential for biolog-
ical activity, with its acetyl ester being inactive. The hy-
droxy function, the pyridine nitrogen and the 3-acyl
substituent may bind to the amino acid background of
the ATP-binding pocket of CDK1.
HO
O
O
H
H
R
(iii)
H
O
N
In order to investigate the importance of the nitrogen as
potential hydrogen bond acceptor function, its basicity
was varied by the introduction of varying electron
releasing substituents in the 3-position instead of the
electron withdrawing 3-acyl function causing lower
basicity and less hydrogen bond acceptor properties.
O
O
O
O
HO
R
Although we changed just one functional group, while
maintaining a hydrogen bond acceptor function, we ob-
served significant shifts in the CDK inhibition profile.
Such characteristics have not been previously observed
for structurally changed classes of CDK inhibitors.1
3
(iii)
R
+
O
N
N
7a-e
The novel CDK-inhibition profiles encourage further
development of specific AD therapeutics or modifying
acting cytostatics.
Scheme 1. Reagents and conditions: (i) PhOCOCl, THF, À20°C; (ii)
PhMgCl, CuI, À20°C; (iii) dioxane/HClO4 (5%), rt.
The N-phenoxycarbonyl-1,4-dihydropyridines 6a–e
were prepared from corresponding commercially availa-
ble pyridine compounds 5a–e by primary acylation using
equimolar amounts of phenylchloro formate at low tem-
perature (À20°C) in dried tetrahydrofurane to interme-
diate acylpyridinium compounds and consequent
regioselective 4-phenylation reaction using equimolar
amounts of phenylmagnesium chloride and copper(I) io-
dide as catalyst.9,10
3-chloro and 3-bromo 1-aza-9-oxafluorenes 7a,d and
7e11 were yielded after work up and preparative column
chromatography as main products beside small amounts
of pyridine oxidation side products (ꢀ20%), yields of the
3-methoxy and -ethoxy compounds 7b,c11 were compa-
rably poor (20% and 25%) with pyridine compounds
being the main products formed during the primary
cycloaddition procedure.
The resulting pure crystalline 1,4-dihydropyridines 6a–e
with overall yields of 85% were treated with a 1.2M ex-
cess of p-benzoquinone in dioxane containing 5% of per-
chloric acid as catalyst at room temperature. Primary
intermediate tetrahydro-1-aza-9-oxafluorenes were oxi-
dized with excess p-benzoquinone added in each 0.3M
portions until no more tetrahydro-intermediate was
detectable by TLC (Scheme 1). While resulting 3-methyl,
Biological evaluations as a CDK inhibitor indicated no
activity for the 3-methyl compound 7a with an electron
releasing function (+I (inductive)-effect) but without
hydrogen bond binding ability of the 3-substitution
(Table 1). This results suggest that the 3-acyl function
of the previous series 4 is essential for biological activity
with a suggested hydrogen bond acceptor function to
the ATP-binding pocket backbone.

B. Voigt et al. / Bioorg. Med. Chem. Lett. 15 (2005) 823–825
Table 1. CDK inhibition profiles of novel 1-aza-9-oxafluorenes 7a–e
825
In summary, structural variations of just one functional
group in the class of CDK-inhibitory active 1-aza-9-oxa-
fluorenes causes surprising certain shifts in the selectivity
profiles to CDK5/p25 and GSK-3b as well as CDK2/4
and encourages concentration on the development of
different therapeutics against AD and CDK-sensitive
cancer. Consequent variations of the 4-phenyl substitu-
tion will be of interest to improve inhibitory activity
and so strengthen selectivity inhibition profiles.
Compd
IC50 (lM)a
CDK1/B
elbfluorene 4.2 1.1
CDK5/p25 CDK2/E
CDK4/D
Ndb
Nac
>100
>100
80.0 24.3 >100
>100
>100
7a
7b
7c
7d
7e
>100
64.3 16.2 6.3 0.4
>100
>100
70.7 17.6 Nac
5.9 1.2
Nac
>100
>100
3.2 0.3
>100
>100
28.7 4.0
a Means of at least two determinations. CDK inhibition experiments
were carried out as described.9
b Nd, not determined.
c Na, not active.
Acknowledgements
This work was financially supported by the country Sax-
ony-Anhalt within its graduate program to Burkhardt
Voigt.
However, with maintaining hydrogen bond acceptor
function at the 3-position, the 3-methoxy substitution
in 7b with mainly electron releasing effects (ÀI and
+M (mesomeric)-effect) surprises with a loss of CDK1-
inhibition (IC50 = 64.3lM) compared to a 3-acetyl sub-
stitution in 4a, elbfluorene, with IC50 (CDK1) of 4.2lM.
An increase in CDK5/p25 inhibition with IC50 of 6.3lM
was found compared to elbfluorene 4a with some
CDK5/p25 inhibitory activity of 15% observed at
10lM.9 CDK2 remains hardly affected by 7b.
References and notes
1. Huwe, A.; Mazitschek, R.; Athanassios, G. Angew. Chem.,
Int. Ed. 2003, 115, 2170; Huwe, A.; Mazitschek, R.;
Athanassios, G. Angew. Chem., Int. Ed. 2003, 42, 2122.
2. Gu, Y.; Rosenblatt, J.; Morgan, D. O. EMBO J. 1992, 11,
3995.
3. Sherr, C. J. Science 1996, 274, 1672.
An increase in selectivity of CDK-inhibition was found
for the 3-ethoxy derivative 7c with practically no activity
against CDK1, CDK2 and CDK4 and an exclusive
activity against CDK5/p25 with an IC50 value of 5.9lM.
4. Brandeis, M.; Hunt, T. EMBO J. 1996, 5280.
5. Knockaert, M.; Greengard, P.; Meijer, L. Trends Phar-
macol. Sci. 2002, 23, 417.
6. Caricasole, A.; Copani, A.; Caruso, A.; Caraci, F.;
Iacovelli, L.; Sortino, L.; Terstappen, G. C.; Nicoletti, F.
Trends Pharmacol. Sci. 2003, 24, 233.
7. Kunick, C.; Lauenroth, K.; Leost, M.; Meijer, L.; Lemcke,
Th. Bioorg. Med. Chem. Lett. 2004, 14, 413.
8. Brachwitz, K.; Hilgeroth, A. Bioorg. Med. Chem. Lett.
2002, 12, 411.
As CDK5/p25 as well as GSK-3b are interesting targets
for selective inhibitors as potential AD therapeutics we
also investigated the inhibition of GSK-3b12 by 7b,c
and observed IC50 values of 3.9 1.0lM (7b) and
5.2lM 1.9lM (7c) a partly even better inhibitory
activity than against CDK5/p25.
9. Brachwitz, K.; Voigt, B.; Meijer, L.; Lozach, O.; Scha¨ch-
´
tele, Ch.; Molnar, J.; Hilgeroth, A. J. Med. Chem. 2003,
46, 876.
The different electronic effects of the 3-substituents with
(ÀI) and (+M) effects of the methoxy and ethoxy func-
tions in 7b,c and with only electron withdrawing effects
(ÀI and ÀM) of the acetyl function in elbfluorene
mainly influence potential nitrogen binding ability to
the CDK protein backbone and may give a plausible
explanation for the observed shift in the CDK inhibition
profile.
10. Hilgeroth, A.; Brachwitz, K.; Baumeister, U. Heterocycles
2001, 55, 661.
11. NMR and MS data for target compounds 7a–e: Com-
1
pound 7a: H NMR (DMSO-d6) d 9.30 (s, 1H), 8.33 (s,
1H), 7.66–7.57 (m, 3H), 7.50 (d, J = 8.8Hz, 1H), 7.46–7.43
(m, 2H), 6.90 (dd, J = 8.8/2.5Hz, 1H), 6.28 (d, J = 2.5Hz,
1H), 2.18 (s, 3H); MS (EI) m/z 275 (M+). Compound 7b:
1H NMR (DMSO-d6) d 9.34 (s, 1H), 8.25 (s, 1H), 7.61–
7.53 (m, 3H), 7.51–7.49 (m, 2H), 7.49 (d, J = 8.9Hz, 1H),
6.91 (dd, J = 8.9/2.5Hz, 1H), 6.47 (d, J = 2.5Hz, 1H),
3.84 (s, 3H); MS (ESI) m/z 292 (M + H+). Compound 7c:
1H NMR (DMSO-d6) d 9.33 (s, 1H), 8.25 (s, 1H), 7.59–
7.51 (m, 5H), 7.49 (d, J = 8.9Hz, 1H), 6.91 (dd, J = 8.9/
2.5Hz, 1H), 6.51 (d, J = 2.5Hz, 1H), 4.09 (q, J = 7.1Hz),
1.18 (t, J = 7.1Hz, 3H); MS (ESI) m/z 306 (M + H+).
Surprising differences have been found for the 3-halogen
substituted derivatives 7d and 7e: While the 3-chloro
compound 7d was found inactive as CDK inhibitor,
the bromo derivative shows an inhibition profile shift
to CDK2 and CDK4, a set of single CDKs, which are
often commonly inhibited by CDK inhibitors.
1
Compound 7d: H NMR (DMSO-d6) d 9.44 (s, 1H), 8.66
(s, 1H), 7.65–7.64 (m, 3H), 7.57 (d, J = 8.7Hz, 1H), 7.49–
7.47 (m, 2H), 6.96 (dd, J = 8.7/2.5Hz, 1H), 6.24 (d,
J = 2.5Hz, 1H); MS (EI) m/z 295 (M+). Compound 7e: 1H
NMR (DMSO-d6) d 9.45 (s, 1H), 8.57 (s, 1H), 7.67–7.63
(m, 3H), 7.57 (d, J = 8.7Hz, 1H), 7.54–7.51 (m, 2H), 6.97
(dd, J = 8.7/2.5Hz, 1H), 6.33 (d, J = 2.5Hz, 1H); MS (EI)
m/z 339 (M+).
Electronic effects of the halogen substituents may con-
tribute to the shift in the CDK-inhibition profile. The
basicity of the nitrogen in 7d and 7e may be different
compared to the acyl-substitution in 4a, as well as to
the alkoxy-substitution in 7b and 7c, with combined
electron withdrawing (ÀI) and electron releasing (+M)
effects of the halogen atoms. However, the difference
of activity of the chloro and bromo compound remains
an interesting fact, which has to be investigated further.
12. Leost, M.; Schultz, C.; Link, A.; Wu, Y.-Z.; Biernat, J.;
Mandelkow, E.-M.; Bibb, J. A.; Snyder, G. L.; Greengard,
P.; Zaharevitz, D. W.; Gussio, R.; Senderowicz, A. M.;
Kunick, C.; Meijer, L. Eur. J. Biochem. 2000, 267, 5983.