Parallel synthesis of acylsemicarbazide libraries: Preparation of potent cyclin dependent kinase (cdk) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.09.023

Potent cyclin dependent kinase inhibitors were prepared using parallel synthesis methodology. Treating advanced intermediate 2 with a variety of hydrazides in DMSO at 80°C for 30 min gave the desired acylsemicarbazides in good to excellent yield. Several compounds were active against cdk4/D1 and cdk2/E in the low nanomolar range. The SAR indicates a wide variety of substituents are tolerated at the acylsemicarbazide moiety. Potent cyclin dependent kinase inhibitors were prepared using parallel synthesis methodology. Treating advanced intermediate 2 with a variety of hydrazides in DMSO at 80°C for 30 min gave the desired acylsemicarbazides in good to excellent yield. Several compounds were active against cdk4/D1 and cdk2/E in the low nanomolar range. The SAR indicates a wide variety of substituents are tolerated at the acylsemicarbazide moiety.

Full text of DOI:10.1016/j.bmcl.2004.09.023

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5489–5491
Parallel synthesis of acylsemicarbazide libraries: preparation
of potent cyclin dependent kinase (cdk) inhibitors
David A. Nugiel,* Anup Vidwans and Carolyn D. Dzierba
Dupont Pharmaceuticals, PO Box 80336, Wilmington, DE 19880-0336, USA
Received 4 June 2004;revised 3 September 2004;accepted 4 September 2004
Available online 30 September 2004
Abstract—Potent cyclin dependent kinase inhibitors were prepared using parallel synthesis methodology. Treating advanced inter-
mediate 2 with a variety of hydrazides in DMSO at 80°C for 30min gave the desired acylsemicarbazides in good to excellent yield.
Several compounds were active against cdk4/D1 and cdk2/E in the low nanomolar range. The SAR indicates a wide variety of sub-
stituents are tolerated at the acylsemicarbazide moiety.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
new class of inhibitors prepared in an efficient manner
using parallel synthesis.
A fundamental process in biology is the division of cells
mediated by the cell cycle.1 Over expression of the tumor
promoting components or the loss of tumor suppressing
products can lead to unregulated cellular proliferation
and the generation of tumors.2 Cyclin dependent kinases
(cdks) play a key role in regulating the cell cycle machin-
ery.3 An increasing body of evidence has shown a link
between tumor development and cdk-related malfunc-
tions.4 This evidence has led to an intense search for
small molecule inhibitors of the cdk family as an ap-
proach to cancer chemotherapy.5 We recently disclosed
a novel class of potent cdk inhibitors based on the
indenopyrazole core (Fig. 1).6 This report describes a
2. Chemistry
Scheme 1 details the preparation of acylsemicarbazide
containing indenopyrazoles using parallel synthesis.
The preparation of intermediate 1 was described previ-
ously.6 This aniline was selectively protected on the pyr-
azole NH using SEMCl and triethylamine in refluxing
CHCl3 in 78% yield. The aniline was converted to the
corresponding phenylcarbamate using phenylchlorofor-
mate and K2CO3 in 75% yield. Intermediate 2 was our
key building block from which acylsemicarbazide librar-
ies were generated. Treating carbamate 2 with assorted
carbazates in DMSO at 80°C gave good to excellent
yields of the desired acylsemicarbazides. The DMSO
was removed at reduced pressure and the SEM protect-
ing group was removed by dissolving the residue in a 1:1
mixture of EtOH and 4N HCl in dioxane. Heating this
mixture for 30min at 80°C and subsequent purification
by LC/MS gave the desired products in 70–93% yield for
O
OMe
H
NH
O
1
NH
the two steps. H NMR, MS, and HPLC, characterized
each compound.
N
Figure 1. Potent indenopyrazole-based cyclin dependent kinase inhib-
itor. Cdk2/E IC50 = 0.24lM;cdk4/D1 IC 50 = 0.4lM.
3. Results and discussion
Keywords: Cyclin dependent kinase (cdk) inhibitors;Acylsemicarb-
azide;Parallel synthesis.
Our library consisted of 84 compounds that were
screened against cdk2/E and cdk4/D1 at an initial inhib-
itor concentration of 0.03lM to determine preliminary
*
Corresponding author. Tel.: +1 302 885 5018;fax: +1 302 886
4989;e-mail: david.nugiel@astrazeneca.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.023

5490
D. A. Nugiel et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5489–5491
O
Ph
OMe
OMe
NH2
O
NH
O
O
a, b
NH
N
N
N
SEM
O
H
R
N
OMe
N
H
NH
O
O
c, d
NH
N
Scheme 1. Reagents and conditions: (a) SEMCl, Et3N, CHCl3, reflux 78%;(b) PhOC(O)Cl, K 2CO3, acetone, 50°C, 75%;(c) RC(O)NHNH 2, DMSO,
80°C, 70–93%;(d) 4N HCl, dioxane, 80 °C, 91%.
Table 1. List of substituents used to prepare acylsemicarbazide library
activity. Analogs showing a percent inhibition of >50%
had standard six-point IC50 curves determined. The re-
sults of those measurements are presented in Table 2.
NO2
HO
Me
Me
MeO
In general, acylsemicarbazides provide an excellent scaf-
fold for generating inhibitors of both cdk2 and cdk4. A
wide variety of substituents are tolerated in the SAR.
There is a clear preference for substituted aromatic func-
tionality. Branched alkyl groups are tolerated (3a) but a
simple methyl group looses around 5-fold in potency
especially against cdk4. The binding pocket accommo-
dates both electron donating (3f,k,u) and electron with-
drawing groups (3d,i,w) in a similar fashion. These
substituents can be displayed in a variety of patterns
without a significant impact on the compoundÕs potency
(Table 1).
S
3a
OMe
3b
3c
3d
Cl
3e
MeO
HO
HO
MeO
Me
H2N
HO
Cl
3i
3f
NH2
3g
3j
3h
H2N
MeO
N
H
H2N
3k
3l
3m
3n
3o
This compound class is slightly more active against
cdk4 than cdk2. The margin of selectivity is not large,
at best 3-fold in favor of cdk4 inhibition (3h). In one
example the activity profile is reversed and cdk2
inhibition is greater than cdk4 inhibition (3ab). This
compound is also 10-fold less potent than the other
examples in the table. This observation may have more
to do with the desolvation penalty associated with bind-
ing this highly solvated substituent rather than an unfav-
orable interaction with the residues lining the binding
pocket.
OH
Me
OMe
3p
3q
3r
3s
3t
OMe
HO
OH
N
N
MeO
3u
3v
3w
3x
3y
HO
H2N
O
Me
H2N
Table 2 contains cell-based data for compounds that in-
hibit the growth of a transformed human colon cancer
cell line (HCT116). The compounds are active in the
0.03–1lM range. Compounds containing hydrophobic
substituents at the acylsemicarbazide moiety give better
results than compounds with polar substituents. This
observation seems to reflect the compoundÕs ability to
cross the cell membrane and influence cell growth rather
than a measure of the compoundÕs ability to inhibit en-
zyme activity. Most of the compounds taken into the
cell-based assay are quite potent against both cdks
measured (<0.03lM) with little variance between the
strongest and weakest inhibitors.
H2N
3z
3aa
3ab
3ac
3ad
4. Conclusions
We have presented a facile method for preparing a novel
class of cdk inhibitors using parallel synthesis. These
acylsemicarbazides are highly potent against both
cdk2/E and cdk4/D1. The compounds also inhibit the
growth of a transformed human colon cancer cell line.

D. A. Nugiel et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5489–5491
5491
Table 2. Enzymatic and cellular activity for selected compounds
Catherine Burton for providing the cell-based assay
data. In addition, we gratefully acknowledge Dongliang
Zhan for providing assistance with the LC/MS data
analysis.
O
H
R
N
OMe
N
H
NH
O
O
References and notes
NH
N
Compounds
Cdk2/E
IC50, lMa
Cdk4/D1
IC50, lMa
HCT116
IC50, lMa
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3c
0.019
0.013
0.015
0.024
0.020
0.013
0.024
0.036
0.010
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0.13
0.0065
0.009
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0.007
0.007
0.028
0.005
0.016
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.28
NT
0.05
0.04
0.14
0.08
0.12
NT
NT
0.03
0.19
0.07
0.25
NT
1.1
3d
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
0.019
0.053
0.016
0.014
0.024
0.009
0.035
0.033
0.041
0.012
0.012
0.016
0.016
0.037
0.009
0.097
0.013
0.01
NT
NT
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NT
0.06
3s
0.013
0.027
0.024
0.007
0.009
0.009
0.012
0.057
0.005
0.24
3t
3u
3v
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3w
3x
3y
3z
7. The in vitro assays employ cell lysates from insect cells
expressing either of the kinases and subsequently their
corresponding regulatory units. The cdk/cyclin lysate is
combined in a microtiter-type plate along with a kinase
compatible buffer, 32P-labeled ATP at a concentration of
50mM, a GST–Rb fusion protein and the test compound at
varying concentrations. The kinase reaction is allowed to
proceeded with the radiolabeled ATP, then effectively
stopped by the addition of a large excess of EDTA and
unlabeled ATP. The GST–Rb labeled protein is sequestered
on a GSH–Sepharose bead suspension, washed, resus-
pended in scintillant, and the 32P activity detected in a
scintillation counter. The compound concentration, which
inhibits 50% of the kinase activity, was calculated for each
compound.
8. We examined the effect of these compounds on cultured
HCT116 cells and determined their effect on cell-cycle
progression by the colorimetric cytotoxicity test using
sulforhodamine B (Skehan et al. J. Natl. Cancer Inst.
1990, 82, 1107–1112). HCT116 cells are cultured in the
presence of test compounds at increasing concentrations.
At selected time points, groups of cells are fixed with
trichloroacetic acid and stained with sulforhodamine B
(SRB). Unbound dye was removed by washing and protein-
bound dye was extracted for determination of optical
density.
3aa
3ab
3ac
3ad
1.2
0.007
0.01
0.08
0.07
a Values are means of two experiments, NT = not tested. For in vitro
assay conditions see Ref. 7. For cell-based assay conditions see Ref. 8.
A wide variety of substituents are tolerated at the
point of diversity indicating the binding pocket that
accommodates the acylsemicarbazide moiety is
rather large and somewhat lipophilic. We can take
advantage of the promiscuous nature of this binding
pocket to improve the physical properties of these
compounds in an attempt to improve biologically
relevant properties.
Acknowledgements
We gratefully acknowledge Drs. Robert Grafstrom and
Sarah Cox for providing the enzymatic assay data and