A structure-based library approach to kinase inhibitors

Full text of DOI:10.1021/ja9614934

7430
J. Am. Chem. Soc. 1996, 118, 7430-7431
A Structure-Based Library Approach to Kinase
Inhibitors
Thea C. Norman, Nathanael S. Gray, John T. Koh, and
Peter G. Schultz*
Figure 1. Structure of olomoucine and numbering scheme for purine
nucleus.
Howard Hughes Medical Institute
Department of Chemistry
UniVersity of California
Scheme 1. Glycinamide-Based Synthesis of
2-(Acylamino)-6-aminopurines
a
Berkeley, California 94720
ReceiVed May 6, 1996
The purine ring system is a key structural element of the
substrates and ligands of many biosynthetic, regulatory, and
signal transduction proteins including cellular kinases, G
proteins, and polymerases. Consequently, combinatorial librar-
ies based on this scaffold should facilitate the search for
inhibitors of many biomedically significant processes. We have
begun to develop libraries around the purine scaffold in
connection with our efforts to generate selective inhibitors of
the cell cycle kinases. The cyclin-dependent kinases (CDKs)
are the principal regulators of processes such as cell growth,
a
Conditions: (a) 1.1 equiv of NaH, 1 equiv of MeI, DMF; (b) 3
equiv of trifluoroacetic anhydride, CH
equiv of tert-butyl R-iodoacetate, DMF; (d) aqueous K
(e) TFA, 1,4-dimethoxybenzene; (f) 1 equiv of PyBroP, 1 equiv of
p-nitrophenol, 3 equiv of DIEA, CH Cl ; (g) 0.05 M 3, Rink-derivatized
solid support, 0.06 M DIEA, DMF, 37 °C, 12 h; (h) 0.2 M R COCl,
.25 M 4-methyl-2,6-di-tert-butylpyridine, CH Cl , 37 °C, 12 h; (i) 0.25
, DMF/DMSO, 1:1 (v/v), 4 °C, 16 h; (j) CH Cl /TFA/Me
(v/v), rt, 2 h.
2
Cl
2
; (c) 1.1 equiv of NaH, 2
2
CO , MeOH;
3
1
DNA replication, and cell division. In human cells, CDC2 and
CDK2 have been implicated in the control of mitosis and DNA
replication, respectively.2 A number of studies have provided
data that support the importance of these CDKs in human
2
2
,3
1
0
2
2
2
diseases such as cancer4 and restinosis, and have stimulated
,5
6,7
M R NH
2
2
2
2
S
8
an active search for chemical inhibitors of these kinases. While
purine analogs were being screened for inhibition of various
apparatus.11 The purine scaffold is attached to the support by
either a glycinamide installed at C-2 or a hydroxyethyl sub-
stituent installed at N-9. Initial synthetic efforts focused on
preparing a 6-chloropurine derivative bearing an active ester
that could be used to derivatize pins containing an acid-labile
protein kinases, a relatively selective inhibitor, olomoucine
9
(
Figure 1), was identified that competitively inhibits CDK2/
cyclin A with an IC50 of 7 µM.
A comparison of the CDK2 crystal structures containing
bound ATP and bound olomoucine confirms that olomoucine
binds in the adenine binding pocket of CDK2, but its purine
nucleus adopts an entirely different orientation than that
1
2
Rink linker (Scheme 1). The sequence begins with the
regioselective methylation of 1 which affords a separable 7:1
mixture of 9- and 7-methyl-2-amino-6-chloropurine isomers,
10
observed for ATP. In spite of the good shape complementarity
shown by the olomoucine-CDK2 complex, structural variations
at C-6, C-2, and N-9 might be expected to lead to enhanced
affinity and selectivity for CDK2. The coupling of this
structural information with combinatorial methods is an obvious
strategy for optimizing olomoucine’s potency. Herein we apply
this approach to the solid-phase synthesis and screening of
combinatorial libraries based on the purine scaffold found in
olomoucine.
1
3
respectively.
The exocyclic amine is trifluoroacetylated,
alkylated with tert-butyl R-iodoacetate, and the alkylated
trifluoroacetamide is saponified. Acid-catalyzed cleavage of 2
followed by PyBroP (bromotripyrrolidinophosphonium hexaflu-
1
4
orophosphate)-mediated activation of the free acid with
p-nitrophenol (PNP) provides active ester 3 which can be stored
15
indefintely at 4 °C. Coupling of 3 to support-bound free amine
(
1.1 µmol/pin) can be monitored by a quantitative ninhydrin
1
6
procedure and is typically complete within 12 h.
In order to facilitate both the chemical and biological
evaluation of soluble olomoucine analogues, synthesis is
performed in a spatially-separated fashion using Geysen’s pin
The first combinatorial step consists of acylating the exocyclic
nitrogen of 4. Treatment of the purine with a dichloromethane
solution of the acid chloride in the presence of 2,6-di-tert-butyl-
*
To whom correspondence should be addressed.
4
-methylpyridine results in complete coupling after 12 h,
(
(
(
1) Norbury, C.; Nurse, P. Annu. ReV. Biochem. 1992, 61, 441-470.
1
7
providing tertiary amide 5. Reversed-phase HPLC studies
2) Fang, F.; Newport, J. W. Cell 1991, 66, 731-742.
3) Pagano, M.; Pepperkok, R.; Lukas, J.; Baldin, V.; Ansorge, W.;
established that even sterically congested groups can be attached
to the purine scaffold using this protocol. The second combi-
natorial step is the nucleophilic aromatic substitution of chlo-
ropurine 5 by primary and secondary amines. Competitive
Bartek, J.; Draetta, G. J. Cell Biol. 1993, 121, 101-111.
(
4) Kamb, A.; Gruis, N. A.; Weaver-Feldhaus, J.; Liu, Q.; Harshman,
K.; Tavtigian, S. V.; Stockert, E.; Day III, R. S.; Johnson, B. E.; Skolnik,
M. H. Science 1994, 264, 436-440.
(5) Nobori, T.; Miura, K.; Wu, D. J.; Lois, A.; Takabayashi, K.; Carson,
D. A. Nature (London) 1994, 368, 753-756.
6) Simons, M.; Edelman, E. R.; DeKeyser, J.; Langer, R.; Rosenberg,
R. D. Nature 1992, 359, 67-70.
7) Morishita, R.; Gibbons, G. H.; Ellison, K.; Nakajima, M.; Zhang,
L.; Kaneda, Y.; Ogihara, T.; Dzau, V. J. Proc. Natl. Acad. Sci. U.S.A. 1993,
0, 8474-8478.
(11) Geysen, H. M.; Rodda, S. J.; Mason, T. J.; Tribbick, G.; Schoofs,
P. G. J. Immunol. Methods 1987, 102, 259-274.
(
(12) Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790.
(13) This reaction is particularly regioselective for alkylation at N-9 and
typically affords a 5:1 mixture of N-9/N-7 alkylation products (85% yield)
which can be separated by flash chromatography.
(
9
(
8) A recent paper describing a genetic approach to identification of
(14) Coste, J.; Dufour, M-N.; Pantaloni, A.; Castro, B. Tetrahedron Lett.
1990, 31, 669-672.
specific CDK2 inhibitors has appeared: Colas, P.; Cohen, B.; Jessen, T.;
Grishina, I.; McCoy, J.; Brent, R. Nature 1996, 380, 548-550.
(15) The overall yield for this five-step sequence is 32%.
(16) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal.
Biochem. 1981, 117, 147-157.
(
9) Vesely, J.; Havlicek, L.; Strnad, M.; Blow, J. J.; Donella-Deana, A.;
Pinna, L.; Letham, D. S.; Kato, J.; Detivaud L.; Leclerc., S.; Meijer, L.
Eur. J. Biochem. 1994, 224, 771-786.
(17) HPLC analysis was performed using a Rainin C18 column, running
a 40-100% gradient of methanol in water buffered with 0.5% triethylam-
monium acetate (pH 8.0). UV detection of peaks was monitored at either
254 or 310 nm.
(10) Schulze-Gahmen, U.; Brandsen, J.; Jones, H. D.; Morgan, D. O.;
Meijer, L.; Vesely, J.; Kim, S.-H. Proteins: Struct., Funct., Genet. 1995,
2, 378-391.
2
S0002-7863(96)01493-X CCC: $12.00 © 1996 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 31, 1996 7431
Scheme 2. 9-(2-Hydroxyethyl)-Linked Synthesis of
2
Library synthesis begins with the acylation of 9 with
,6-Diaminopurinesa
trifluoroacetic anhydride and is followed by alkylation with the
21
desired alcohol under Mitsonobu conditions to afford trifluo-
roacetamide 10. Subsequent amination is accompanied by
aminolysis of the trifluoroacetamide and provides purines having
the structure 11. Purine alcohol 12 is cleaved from the support
using 90:10 trifluoroacetic acid/water. Six purine analogues
were prepared on aminoalkyl-derivatized resin (0.87 mmol/g)
using this five-step sequence with overall isolated yields of 75-
8
5%. Following this, a small library of 16 alkylated aminopu-
rines was prepared on pins using primary and benzylic alcohols.
Again HPLC analysis of the cleaved material revealed few, if
any, side products.
As a first step in the combinatorial optimization of olomou-
cine, we targeted the C-6 position of the purine core for
2
2
substitution with a wide variety of amines.
Using the
glycinamide-linked purine scaffold, a library of 348 purine
derivatives was prepared using 5 acid chlorides and 58 amines
plus 58 aminated pins bearing no acyl group. Evaluation of
the library was carried out using a microtiter-based solution-
a
Conditions: (a) 1.1 equiv of 2-(benzyloxy)ethanol, 10 mol %
p-TsOH, CH
2
Cl
2
, -10 °C to rt, 6 h; (b) H
2
, 10% Pd/C, EtOAc, rt, 24
, 1 equiv of
23
24
phase assay for protein kinase activity which identified
CDKZ inhibitors containing meta- and para-substituted ben-
zylamines that appeared to be more active than olomoucine.
Solution-phase synthesis and characterization of one such
derivative, 2-((2-hydroxyethyl)amino)-6-((4-methoxybenzyl)-
h; (c) 1 equiv of 2-amino-6-chloropurine, 2 equiv of PPh
3
diethylazodicarboxylate (DEAD), THF, -10 °C to rt, 12 h; (d) (i) 1
equiv of 1:1 2 N NaOH dioxane, rt, 12 h, (ii) 2 equiv of pentafluo-
rophenol, 1.1 equiv of diisopropylcarbodiimide, DMF, 0 °C to rt, 6 h;
iii) 0.05 M PFP ester, aminoalkyl support, 0.06 M DIEA, DMF, 37
C, 12 h; (e) (i) 0.2 M trifluoroacetic anhydride, 0.30 M 4-methyl-
,6-di-tert-butylpyridine, CH
PPh
0 °C, 12 h; (g) TFA/H
(
°
2
9
amino)-9-(isopropylamino) provided an inhibitor having an IC50
1
2
Cl
2
, 37 °C, 4 h, (ii) 0.2 M R OH, 0.4 M
(600 nM) more than an order of magnitude lower than that
measured for oloumucine (7 µM).
2
3
2
, 0.2 M DEAD, THF, -10 °C to rt, 6 h; (f) 0.25 M R NH , DMSO,
7
2
O, 90:10 (v/v), rt, 1 h.
Using the acylation and Mitsonobu chemistry described
above, we are currently constructing larger libraries (>5000)
in which a variety of structures are appended to the amino group
at C-2. New strategies are also being developed to introduce
N-9 substituents in a combinatorial format and to construct
macrocycles between these substituents and those at C-2. The
iteration of library synthesis with structural analysis of the
optimized leads should provide an effective strategy for the
development of more potent and selective inhibitors of CDK2.
In addition, libraries containing purine derivatives may prove
useful in the search for inhibitors of a large number of cellular
processes.
1
deacylation of R is minimized by performing the amination in
a 1:1 mixture of DMF/DMSO (0.25 M in amine) at 4 °C. After
cleavage from the support using 80:15:5 dichloromethane/
trifluoroacetic acid/dimethyl sulfide, this four-step sequence
results in purine derivatives having the general structure 6.
For characterization purposes, seven purine analogues were
prepared on Rink-derivatized resin (0.59 mmol/g) and evaluated
17
by reversed-phase HPLC analysis.
The compound corre-
sponding to the major peak18 was isolated, and the structure of
the purine derivative was verified by high-resolution spectros-
1
19
copy ( H NMR and FAB-MS). Following this, a small library
of 36 purine derivatives was prepared on pins using six acid
chlorides and five amines, plus five aminated pins having no
acyl group. The entire library was evaluated by reversed-phase
HPLC which in all cases indicated that very few, if any, purine-
containing side products were produced during the sequence.
UV analysis of material cleaved from pins (289 nm, ꢀ ) 12 000
Acknowledgment. This work was supported by the Director, Office
of Health Effects Research, of the U.S. Department of Energy under
Contract No. DE-AC03-76SF00098. P.G.S. is a Howard Hughes
Medical Institute Investigator. T.C.N. is supported by a NSF post-
doctoral fellowship (Grant CHE9301146); N.S.G. is supported by a
NSF predoctoral fellowship; J.T.K. is supported by an American Cancer
Society postdoctoral fellowship. We thank Professor Sung-Hou Kim
for providing us with crystallographic information, Professor David
Morgan for the generous gift of activated CDK2/cyclin A and Dr.
Andrew Bray of Chiron Mimotopes for helpful discussions and for the
supply of derivatized pins.
-
1
-1
M
cm ) indicated a 30-75% overall yield for the solid-phase
chemistry.
In order to expand the chemistry that can be carried out at
the C-2 amino group to include alkylation reactions, a strategy
was devised to attach the purine core to the support through
N-9. Initial efforts to load 2-amino-6-chloro-9-(2-hydroxyeth-
yl)purine to a solid support functionalized with an acid-labile
dihydropyran linker resulted in the exclusive attachment of the
purine to the linker via the exocyclic amine at C-2. For this
reason, solution-phase chemistry was developed to attach the
purine core to the tetrahydropyranyl linker prior to loading
Supporting Information Available: Experimental procedures and
analytical data for the synthesis and chracterization of the compounds,
description of the solid-phase chemistry depicted in Schemes 1 and 2,
listing of the building blocks used in the 2-(acylamino)-6-aminopurine
library, and analytical evaluation of the 2-(acylamino)-6-aminopurine
library (10 pages). See any current masthead page for ordering and
Internet access instructions.
(
Scheme 2).20 The modified purine is then attached to the solid
JA9614934
support by reacting the aminoalkyl-derivatized pins (1.1 µmol/
(
21) Mitsonobu, O. Synthesis 1981, 1-28.
pin) with the pentafluorophenyl ester corresponding to 8.
(22) Amine building blocks included both primary and secondary amines,
substituted benzylamines, heteroaromatic amines, amino acids, and amino
alcohols.
(
(
(
18) In most instances only a single peak was detected.
19) Yields for the four-step sequence on resin ranged from 60 to 85%.
20) Dihydropyran 7 was prepared in three steps and 44% overall yield
(23) Buxbaum, J. D.; Dudai, Y. Anal. Biochem. 1988, 169, 209-215.
(24) Crude lysates containing activated CDK2/cyclin A (∆171, modified
with His-6) were obtained from Professor David O. Morgan and purified
to homogeneity.
from commercially available 3,4-dihydro-2H-pyran-2-carboxylic acid,
sodium salt.