The Discovery of a New Structural Class of Cyclin-Dependent Kinase Inhibitors, Aminoimidazo[1,2-A]Pyridines

Home , Apoptosis, CDK inhibitor, Cyclin, Cyclin A, Cyclin B, Cyclin E

Molecular Cancer Therapeutics 1

The discovery of a new structural class of cyclin-dependent kinase inhibitors, aminoimidazo[1,2-a]pyridines

Chafiq Hamdouchi,1 Heather Keyser,1 Introduction Elizabeth Collins,1 Carlos Jaramillo,2 2 1 Cyclin-dependent kinases (CDKs) and their cyclin partners Jose Eugenio De Diego, Charles D. Spencer, are key players in regulating the entry into, passage Jack Alan Dempsey,1 Bryan D. Anderson,1 1 1 through, and exit from the cell cycle (1–8). Elevated levels Tillie Leggett, Nancy B. Stamm, of CDK2 have been correlated with a poor prognosis in Richard M. Schultz,1 Scott A. Watkins,1 1 1 1 patients, presumably due to uncontrolled proliferation Kim Cocke, Stephanie Lemke, Teresa F. Burke, which is the basis of neoplastic disease (9–13). CDK2 has Richard P. Beckmann,1 Jeffrey T. Dixon,3 3 3 been shown to phosphorylate Rb, p107, p130, p27, cdc25A, Thomas M. Gurganus, Nancy B. Rankl, DP1, cdh1, cdc6, and orc1 (14–18). Much of this binding Keith A. Houck,3 Faming Zhang,1 Michal Vieth,1 2 1 specificity comes from the cyclin partner for CDK2, either Juan Espinosa, David E. Timm, cyclin E or cyclin A, which is required for catalytic activity Robert M. Campbell,1 Bharvin K. R. Patel,1 and 1 (19). Overexpression of cyclin E drives cells into S phase Harold B. Brooks (20, 21). When cyclin A activity is blocked by a peptide 1 Lilly Research Laboratories, Eli Lilly and Company, Lilly inhibitor, selective apoptosis is observed in cancer cells Corporate Center, Indianapolis, IN; 2 Centro de Investigacio´n Lilly, which do not retain a functional Rb protein (22). As a result, Madrid, Spain; and 3 Eli Lilly and Company—Sphinx Labs, there is growing interest in CDK2 as a target for the Research Triangle Park, NC treatment of neoplasia (23). Because of the critical role of the CDKs in the regulation of the cell cycle and the observed expression/activity pattern Abstract in most human cancers, considerable effort has been The protein kinase family represents an enormous oppor- focused on the development of small molecule inhibitors tunity for drug development. However, the current (24–26). However, the number of structural classes that act limitation in structural diversity of kinase inhibitors has as CDK inhibitors is limited, and most of them derive from complicated efforts to identify effective treatments of relatively nonspecific protein kinase inhibitor scaffolds such diseases that involve protein kinase signaling pathways. as staurosporines (27–29), flavonoids (29), indigoids (30), We have identified a new structural class of protein serine/ paulones (31), and purines (32). Flavopiridol (29, 33), a threonine kinase inhibitors comprising an aminoimi- flavonoid derived from an indigenous plant from India, was dazo[1,2-a]pyridine nucleus. In this report, we describe the first CDK modulator tested in clinical trials. Early-phase the first successful use of this class of aza-heterocycles to trials have shown activity in some patients with non- generate potent inhibitors of cyclin-dependent kinases that Hodgkin’s lymphoma and renal, prostate, colon, and gastric compete with ATP for binding to a catalytic subunit of the carcinomas (34). Indirubin, an indigoid active ingredient of protein. Co-crystal structures of CDK2 in complex with a traditional Chinese recipe, was the first example of a CDK lead compounds reveal a unique mode of binding. Using inhibitor used to treat human chronic myelogenous leuke- this knowledge, a structure-based design approach di- mia (CML). Although considerable efforts are still devoted rected this chemical scaffold toward generating potent to these chemical families and their structurally related and selective CDK2 inhibitors, which selectively inhibited analogues, the identification of new structural classes of the CDK2-dependent phosphorylation of Rb and induced protein serine/threonine kinase inhibitors remains highly caspase-3-dependent apoptosis in HCT 116 tumor cells. desirable. This would offer new (potentially more desirable) The discovery of this new class of ATP-site-directed selectivity profiles and new physiochemical properties. protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, Herein we report that the aminoimidazo[1,2-a]pyridine provides the basis for a new medicinal chemistry tool to be scaffold represents a new structural class of protein serine/ used in the search for effective treatments of cancer and threonine kinase inhibitors. Compounds represented by other diseases that involve protein kinase signaling path- structure 1 were demonstrated for the first time to potently ways. [Mol Cancer Ther. 2004;3(1):1–9] inhibit CDKs by competing with ATP for binding to a catalytic subunit of the protein. Moreover, we report the identification of structural features required for kinase inhibition, co-crystal structures of CDK2 in complex with Received 2/21/03; revised 10/7/03; accepted 10/23/03. initial leads, and evidence for a selective inhibition of The costs of publication of this article were defrayed in part by the CDK2 in tumor cells. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods Requests for Reprints: Chafiq Hamdouchi, Lilly Research Laboratories, Chemistry Eli Lilly and Company, Discovery Chemistry and Cancer Research, Lilly Corporate Center, DC: 0540, Indianapolis, IN 46285. Phone: (317) In general, two key synthetic strategies were devised for 276-9582; Fax: (317) 277-3652. E-mail: [email protected] access to this imidazo[1,2-a]pyridine scaffold. The first

strategy is characterized by its highly convergent nature been equilibrated with 50 mM HEPES (pH 7.5), 10% and use of the core 6-iodo-2-(trifluoroacetamido)imi- glycerol, 1 mM DTT, 0.1 mM Pefablock SC (Boehringer- dazo[1,2-a]pyridine (35), as a building block. The two key Mannheim), and 0.1 mM Na3VO4. The enzyme was eluted steps that have led to the success of this approach were a with a 0–1.0 M NaCl gradient. The column fractions were halogen metal exchange on the 6-iodo-2-(trifluoroacetami- assayed for kinase activity as detailed below, pooled do)imidazo[1,2-a]pyridine mediated by isopropyl magne- together, and then diluted to a final NaCl concentration sium bromide and the subsequent lithiation with t-BuLi of 250 mM. This material was applied to a hydroxyapatite directed at the 3-position by a trifluoroacetamide group. column that had been equilibrated with 25 mM HEPES The second strategy is linear in which the key steps are the (pH 7.5), 1 mM DTT, 0.1 mM Na3VO4, and one EDTA free, use of microwave focused irradiation that allowed us to Complete tablet per 400 ml of buffer. The enzyme was overcome the difficulties of the alkylation of 2-chloro- eluted with a 0–500 mM potassium phosphate gradient pyridines and the one-step procedure for the generation of (pH 7.50); the active cyclin E-CDK2 enzyme fractions were 2-aminoimidazo moiety from the corresponding 2-chlor- assayed as indicated below. Peak fractions were then opyridinium salts. The synthetic details will be described pooled and frozen at 80jC. elsewhere. Kinase Assays (CDK2, CDK1, CDK4, GSK3b, CAMKII, Crystallization of hCDK2 PKA, PKC-b) CDK2 was cloned and purified as described previously All reactions were run in 100 Al containing 4% DMSO (36). The protein was concentrated to 10 mg/ml using an for 60 min at room temperature. Variable concentrations Amoco stir cell system. Fresh 10 mM DTT was added just of inhibitor were prepared in 40% DMSO and diluted 10- before crystallization. Crystals were grown in a hanging fold to their final concentration in the reaction. The cyclin drop plate by mixing 10 mg/ml CDK2 at a 1:1 (v/v) ratio D1/CDK4 assay conditions were 35 mM HEPES (pH 7.0), with a solution containing 20% (w/v) PEG-3000, 100 mM 33 10 mM MgCl2, 300 AM ATP, 1 ACi P-g-ATP, 200 AM ING HEPES (pH 7.5), 200 mM NaCl. Orthorhombic crystals of peptide (amino acid residues 246–257 from human Rb dimensions 200 200 200 mm grew within 2–6 days. protein), and 2.19 Ag cyclin D1-CDK4 enzyme. The cyclin X-ray Crystallography Data Collection and Processing E/CDK2 assay conditions were 50 mM HEPES (pH 7.0), X-ray diffraction data were collected at 170jCwitha 33 10 mM MgCl2, 300 AM ATP, 0.5 ACi P-g-ATP, 200 AM ING Mar CCD detector at the IMCA beam line ID-17 at the peptide, and 0.21 Ag cyclin E-CDK2 enzyme. The cyclin Advanced Photon Source in Argonne National Laborato- B/CDK1 assay conditions were 50 mM HEPES (pH 7.5), ries. Data were integrated and reduced using the program 33 10 mM MgCl2,1mM EGTA, 100 AM ATP, 2 ACi P-g-ATP, HKL2000 (37). The crystals belong to space group P212121 40 AM Histone H1 (Invitrogen), and 0.31 ng cyclin B-CDK1 ˚ ˚ with unit cell dimensions of 52.80 A (a), 72.11 A (b), and enzyme (New England Biolabs, Beverly, MA). The PKA ˚ 238.57 A (c). assay conditions were 65 mM HEPES (pH 7.5), 10 mM Structure Solution and Refinement MgCl2,0.12mM CaCl2,3mM DTT, 20 AM ATP, 0.5 ACi The structure was solved by molecular replacement and 33P-g-ATP, 85 Ag of Lys-rich histone (Worthington refined by the CNS program. Sequential model building Biochemicals, Lakewood, NJ), and 6.5 ng PKA enzyme processes were carried out in the graphics program, (Sigma-Aldrich, St. Louis, MO). The CAMKII assay QUANTA98 (38). The final R factor for all data is 24.6%, conditions were 75 mM HEPES (pH 7.5), 10 mM MgCl2, 33 Rfree is 28.0%. The final structure contains 2398 non- 5mM CaCl2,2Ag calmodulin, 30 AM ATP, 1 ACi P- hydrogen protein atoms, 110 water molecules, and 27 g-ATP, 50 AM Autocamtide-2 (BioMol, Plymouth Meeting, compound atoms. All residues are in the most favorable PA), and 1.95 ng calcium calmodulin kinase II enzyme conformation in a Ramachandran plot. (Calbiochem, San Diego, CA). The GSK3h assay condi- Cyclin E-CDK2 Enzyme Purification tions were 150 mM 4-morpholinepropanesulfonic acid Human cyclin E and CDK2 were isolated from a cDNA (MOPS; pH 7.0), 37.5 mM MgCl2, 600 AM ATP, 0.5 ACi 33 library prepared from a human colon carcinoma cell line by P-g-ATP, 50 AM KRREILSRRPpSYR peptide (AnaSpec, PCR amplification using sequence-specific primers. Each Inc., San Jose, CA), 0.09% Triton X-100, and 12 ng GSK3h gene was subcloned into pVL1393 (Invitrogen, Carlsbad, CA). enzyme. The PKC-a,h,q,g assay conditions were 36 mM 33 Recombinant baculovirus stocks expressing cyclin E and HEPES (pH 7.4), 5 mM MgCl2,30AM ATP, 0.5 ACi P- CDK2 were then isolated by transfecting the resultant g-ATP, 1.5 AM RFARKGSLRQKNV peptide (SynPep, plasmids into Sf9 cells along with wild-type AcMNPV Corp., Dublin, CA), 0.1 mM DTT, 0.03% Triton X-100, DNA (Invitrogen). The E-CDK2 enzyme complex was and 10.5, 23, 24, and 147 ng, respectively, for the PKC- prepared by co-expression of the wild-type human cyclin a,h,q, and g enzymes. For cyclin B/CDK1, the reaction E and CDK2 genes in baculovirus-infected insect cells. was terminated with an equal volume of 25% trichloro- After homogenization in 50 mM HEPES (pH 7.5), 320 mM acetic acid, mixed with 25 Ag of BSA carrier, filtered sucrose, 1 mM DTT, 1 mM Na3VO4,2mM EGTA, and 1 through a glass fiber filter plate (Millipore Multiscreen-FC Complete tablet (Roche Diagnostics Corp., Indianapolis, Plate), and washed twice with 10% trichloroacetic acid. IN) per 50 ml of buffer, the material was centrifuged at For the cyclin/CDK, GSK3h, PKC, and CAMKII assays, 35,000 g for 1 h. The supernatant was applied to a Porous the reaction was terminated with an equal volume of 10% Q column (Applied Biosystems, Foster City, CA) that had phosphoric acid, filtered through a phosphocellulose filter

plate (Millipore Multiscreen-PH Plate), and washed twice dissolved in DMSO at 10 mM, and a series of 2-fold with 0.5% phosphoric acid. Microscint 20 (Perkin Elmer, dilutions was made in RPMI 1640. Twenty-microliter Boston, MA) was added to each well, and the plate was aliquots of each concentration were added to triplicate counted on a Packard Top Count scintillation counter. wells. Plates were incubated for 72 h at 37jCina Ki Determination humidified atmosphere of 5% CO2-in-air. In some experi- app The Morrison equation was used to determine the Ki at ments, we analyzed the effect of exposure time on app 0.0626–1.0 mM ATP (39). A plot of Ki versus [ATP] can be antiproliferative activity. In this case, we pulsed the cells used to distinguish between a competitive and noncompet- with compound for either 1, 4, 8, 24, or 72 h. The cells itive behavior, (39). Fitting the data to the competitive were then washed three times after drug exposure with inhibition, the below equation yields an estimate of the media and reincubated for a total of 72 h from the time of slope and a P value for the slope. A P value for the slope of initiation of compound treatment. Following incubation less than 0.05 was used as the criterion for competitive of plates, 10 Al of stock MTT solution was added to all inhibition. wells of an assay, and the plates were incubated at 37jC for 45 min. Following incubation, 200 Al of DMSO was added to each well. Following thorough formazan app ATP solubilization, the plates were read on a Dynatech K ¼ Kið1 þ½ATP=Km Þ i MR600 reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. The IC50’s are reported in Molecular Modeling Table 2. Modeling of inhibitors to the ATP site of CDK2 Cell Cycle Effect of Imidazopyridine on HCT116 protein from the staurosporine complex (40) used Log phase HCT 116 cells were seeded out using RPMI automated DOMCOSAR (41, 42) methodology for dock- 1640 + 10% FBS at 5 105 cells per T-75 flask, and the ing mode determination. The general docking protocol cells were allowed to attach for 24 h at 37jC. The and potential functions employed in the CHARMm- compounds were added to the cells and incubated for an based docking algorithm (CDOCKER) are described in additional 24 h. After incubation, the cells were trypsi- previous papers (43, 44). In this work, we use a var- nized and then centrifuged at 300 g for 5 min. The iation of the ‘‘short annealing’’ schedules described in trypsin was removed, and the cell pellet was washed with detail previously (41). The basic strategy involves 1 PBS, followed by centrifugation at 300 g for 5 min. generation of several initial ligand orientations in the The supernatant was discarded, and the pellet was target protein’s active site followed by MD-based resuspended in 0.5 ml of PBS. Ice-cold 70% ethanol simulated annealing, and finally refinement by minimi- (4.5 ml) was added to the cells while they were being zation. The knowledge of the binding allowed using vortexed; the cells were then held at 20jC for 2–24 h. restrained, automated docking of multiple compounds The ethanol-fixed cells were centrifuged at 300 g for using N1 to NH of Leu83 distance restraint of 3 A˚ and 5 min, the supernatant was removed, and the cell pellet C2 amino group to carbonyl oxygen of Leu83 distance was washed with 1 PBS. Following another centrifuga- restraint of 3 A˚ . tion at 300 g for 5 min, the PBS was removed, and each Nuclear Magnetic Resonance Methods sample pellet was mixed with 1 ml of staining solution Nuclear magnetic resonance (NMR) spectra were ac- (0.1% Triton X-100 [Sigma], 0.2 mg/ml DNase-free RNase quired on a Bruker Avance 500 spectrometer in acetone at A [Sigma], 20 Ag/ml propidium iodide [Molecular Probes] different temperatures (233–323 K) with a broad band in PBS). The cells were then analyzed on a Coulter Epics inverse probe. Proton chemical shifts were referenced to the Flow Cytometer. residual solvent signal at 2.05 ppm. The assignment of Western Blot Analysis of CDK2-Dependent Rb proton resonances was achieved through the combination Phosphorylation 1 of H, 2D correlated spectroscopy, heteronuclear single Human colon carcinoma HCT 116 cells were used in this quantum correlation spectroscopy, nuclear Overhauser study. Cells were maintained in DMEM high glucose enhancer spectroscopy, heteronuclear multibond correla- medium (Life Technologies) supplemented with 10% fetal tion spectroscopy experiments. bovine serum, 0.1 mM non-essential amino acids (NEAA), In Vitro CytotoxicityAssay 1.0 mM Na pyruvate, and 10 mM HEPES. Cells were plated We used the MTT colorimetric assay to measure cell at 4 105 cells in 3 ml volume/well in a six-well plate, and cytotoxicity. The NCI-H460 human lung carcinoma and allowed to attach overnight. Compounds were diluted in HCT 116 human colon carcinoma cell lines were pur- medium at 1,2,or3 IC50, while DMSO was used as a chased from the American Type Culture Collection control. Cells were harvested 24 h after treatment by (Rockville, MD). The cells were cultured in RPMI 1640 washing twice in 3 ml/well cold PBS containing 2 mM containing L-glutamine and 25 mM HEPES buffer and Na3VO4 while rocking. Fifty microliters of radioimmuno- supplemented with 10% dialyzed fetal calf serum. The precipitation assay (RIPA) buffer were added, and cells cells were seeded at 1000 cells/180 Al culture medium per were collected by scraping. To clear the lysate, cells were well in 96-well flat-bottom tissue culture plates 24 h before spun down at 4jC for 10 min, and the supernatant was addition of test compounds. Compounds were initially collected in another tube. Protein concentrations were

Figure 1. A, general structure of the chemical platform. B, two-dimensional representation of the binding mode of compounds 1a and 1b with CDK2. C, chemical structures of imidazo[1,2-a]pyridine-based inhibitors of CDKs disclosed in this manuscript (compounds 1a – 1d).

measured for each lysate by combining 2 Al of sample, 48 Al plates were processed for caspase-3 activity (45). After of 1 PBS, and 200 Al Pierce BCA kit’s reagent A + B (1:50), subtracting the background reading, caspase-3 activity was incubating at 37jC for 30 min, and then measuring in an expressed as a percentage increase compared to untreated ELISA reader at 562 nM. control cells. Samples for electrophoresis and Western blotting were Coordinates heat denatured by incubating at 100jC for 5 min; 25 Alof Coordinates and structure factors of 1a and 1b will be each sample were loaded on 10% Tris-glycine gels. A deposited with the Protein Data Bank. Panther semi-dry electroblotter (Owl Scientific, Portsmouth, NH) was used to transfer gels onto polyvinylidene difluoride membrane at 400 mA for 1 h using Owl buffers. Results and Discussion Membranes were blocked in 6% milk, 0.1% Tween 20 Initial screening of our corporate libraries led to the (Sigma) in Tris-buffered saline for 1 h at room temperature. identification of a group of benzimidazoles as inhibitors of Primary antibody solution (10 ml/blot) was added and several kinases in the micromolar range. This finding, j rocked overnight at 4 C. Blots were washed 3 times for coupled with our early work and understanding of the 10 min each in between the primary and secondary medicinal chemistry link between benzimidazoles and the antibody incubations. The T356 antibody was raised in other aza-heterocycles (35, 46–48), inspired the foundation rabbit to the murine ETERT(PO3)PRK peptide. The total and of the idea of evaluating aminoimidazo[1,2-a]pyridine as a antiphospho-S780 Rb antibody were purchased from Cell potentially novel ATP-competitive inhibitor scaffold. Signaling (Beverly, MA). Pico-Super Signal (Pierce Biotech- Focusing on CDK2 as a target, we systematically explored nology, Rockford, IL) was added for 3 min, and the image the imidazopyridine nucleus based on the several known was captured for 4 min using a Fluor-S Imager (Bio-Rad, ATP-competitive inhibitor classes (25, 27, 31, 32, 49) and Hercules, CA). Band intensity was quantitated using docking experiments (41, 42). We also made an initial Quantity One software (Bio-Rad). Bands were normalized presumption that the 3-position of aminoimidazo[1,2-a] to the actin control. pyridine (Fig. 1) should carry a group that participates Caspase-3 ActivityAssay in the internal hydrogen bond with the amine hydrogen Caspase-3 activity assay was carried out as described (30). In turn, this would hold the second hydrogen in a before (45). Briefly, HCT 116 tumor cells were plated at syn position with the nitrogen at the 1-position, favoring 10,000 cells/well in a 96-well plate. Following overnight an interaction with the protein backbone. This assumption incubation, the cells were treated with compound for either was tested through both SAR studies and X-ray crystal- 24, 48, or 72 h. At the end of the incubation period, the lographic analysis. Initial leads against CDK2 were

compounds 1a (IC50 = 0.32 AM) and 1b (IC50 = 0.12 AM). In solution, these compounds bind in the ATP binding pocket based on their competitive behavior with respect to ATP (Fig. 2). The crystal structures of compounds 1a and 1b bound to the inactive form of human CDK2 (50, 51) were solved (Fig. 3) to reveal the basis for the binding behavior. Inhibitors 1a and 1b were found to occupy the ATP binding site of CDK2 as shown in Fig. 3. Compound 1a shows a unique binding mode with face-to-face internal stacking of aromatic rings (Fig. 3A). Both imidazopyridines share similar interaction with the CDK2 protein. The hydrogen bonding interactions with the hinge backbone, common to the majority of ATP site kinase inhibitors,ispresent.Inparticular,theN-1ofthe

Figure 2. Competitive inhibition of cyclin E/CDK2 by 1a and 1b. The K i imidazopyridines acts as a hydrogen bond acceptor with F F for 1a was 386 9nM (open triangles). The K i for 1b was 87 42 nM the backbone amide NH of Leu83 (heavy atom distance of (closed circles). Data were fit using Sigma Plot 2000 (SPSS Inc., Chicago, 3.26 A˚ ), and the amino group hydrogen donates a IL) to a linear equation and the K is the Y-intercept. Error bars, SE to the fits. i hydrogen bond to the backbone carbonyl oxygen of

Figure 3. A, crystal structure of imidazopyridine 1a bound to CDK2. The compound occupies the space of the ATP pocket. Atoms are colored as follows: N, blue;O,red;F,light blue; and the C atoms of the inhibitor are green and those of the protein are gray. Hydrogen bonds between compound 1a and protein are shown as dashed lines. B, crystal structure of imidazopyridine 1b bound to CDK2. Atoms are colored as follows: N, blue;O,red;F,light blue; and the C atoms of the inhibitor are yellow and those of the protein are gray. C, superposition of imidazopyridine 1a to ATP bound to CDK2. Atoms are colored as follows: N, blue;O,red;F,light blue; and the C atoms of the inhibitor are green and those of ATP are magenta. The protein backbone is shown as a tube in gray.

Table 1. Enzyme inhibition data of imidazopyridines (IC50, AM)

Compound E/CDK2 D1/CDK4 B/CDK1 PKA-a PKC-bII CAMKII GSK3-b no. IC50 (AM) IC50 (AM) IC50 (AM) IC50 (AM) IC50 (AM) IC50 (AM) IC50 (AM)

1a 0.324 5.221 0.326 >20 19.5 3.39 1b 0.122 0.947 0.057 9.57 1.42 0.48 1c 0.026 0.786 0.105 5.54 2.29798 >20 5.9 1d 0.028 0.464 0.143 6.34 3.26201 3.89 >20

Note: Compound 1d, potent inhibitor of CDK2 (IC50 =28nM) and a weaker inhibitor of CDK4 and CDK1 (IC50 = 464 and 143 nM), was tested against a panel of representative kinases and found to be highly selective: GSK3h (>500-fold), CAMKII, PKA, PKC-a,h,q,g (>100-fold).

Leu83 (distance of 2.80 A˚ ). In addition, there is a CDK inhibitor scaffolds have demonstrated poor selectiv- hydrogen bonding interaction between the carbonyl oxy- ity when tested against GSK3h. Here our optimized lead, gen and the backbone NH of Asp145 (heavy atom imidazopyridine 1d, did not show any detectable effect on distance of 3.30 A˚ ). The side chain of Asp145 is 2.7 A˚ GSK3h (Table 1). from one of the aromatic fluorines, which creates These compounds inhibit proliferation in HCT 116 cells speculation of the potential protonation of Asp145 side in tissue culture as determined by an MTT assay (Fig. 4) chain. The difference in binding geometries between 1a (54). This effect is dependent on the drug exposure time as and 1b represents an interesting twist in the competition shown in Table 2 for HCT 116 cells. To demonstrate that the between intra- and intermolecular interactions. The inhibition of growth is a result of target inhibition, we took stacked pose exhibited by 1a has an internal hydrogen advantage of the T356 in the retinoblastoma protein (Rb), bond between the amino group and the carbonyl oxygen which is selectively phosphorylated in vitro by CDK2 (16). while the aromatic rings show face-to-face aromatic In contrast, the S780 site in Rb is selectively phosphorylated stacking. The distance between ring centroids of 4.5 A˚ is by CDK4 in vitro (16, 55). Fig. 5A shows that the T356 site on the low end of stacking between aromatic side chains phosphorylation is inhibited by 1d, whereas the S780 site is in proteins as described by Burley and Petsko (52, 53); left unperturbed at concentrations where cell cycle arrest is however, in the extended pose of 1b, one of the aromatic observed. As shown in Fig. 5A, compound 1d shows rings rotates by 90j to fill a hydrophobic space in the ATP significant inhibition of the CDK2-dependent phosphory- binding pocket, increasing the distance between aromatic lation of T356 on Rb at 24 h, with little or no effect on rings in 1b to 7.5 A˚ . 1H NMR studies of free imidazopyr- CDK4-dependent phosphorylation of S780 on Rb. This idines usually show two sets of signals, indicating the effect is not a consequence of Rb degradation or expression existence of a slow conformational exchange in solution, owing to the restricted rotation around the sp2-sp2 imidazopyridine C3-carbonyl carbon bond. In the case of compound 1b, the rotamer ratio at 253 K is 72:28 as determined by integration. Notably, for the minor rotamer, imidazopyridine H5 appears upfield (7.79 ppm) relative to the major rotamer (10.20 ppm). This large shielding can be attributed to the anisotropic effect of the difluorophenyl ring at H5 position, which exclusively occurs for the stacked pose. Thus, NMR data reveal that the extended pose is also the preferred conformation in solution for 1b. X-ray crystallographic analysis, prelimi- nary SAR study, conformational analysis, and docking studies furnished a number of guidelines for analogue design. On the basis of this analysis, compound 1d was designed, having a key methoxy group at the para position, and was found to be a potent and selective inhibitor of CDK2 (IC50 = 0.028 AM). Compound 1d is also a weaker inhibitor of CDK4 and CDK1 (IC50 = 464 and 143 nM). Compound 1d has also shown high selectivity when Figure 4. Antiproliferative effects of compound 1d on HCT 116 cells F tested against a representative set of other serine/ after 24 h. The IC50 for 1d against HCT 116 was 0.47 0.04 AM (closed threonine kinases: CAMKII, PKA, PKC-a,h,q,g (>100-fold). circles). Data were fit using Sigma Plot 2000 (SPSS) to a four-parameter Hill equation. Dashed line, mean for the untreated controls; dotted lines, Of interest is the remarkable selectivity that was achieved FSD of the controls. Two sets of points, data collected on separate days. with respect to GSK3h (>500-fold). Most of the reported Error bars, SD for the triplicates from a single day’s experiments.

Table 2. Effect of incubation time on the IC50 for antiproliferative effects on HCT 116

Compound no. IC50 (AM) after 1 h IC50 (AM) after 4 h IC50 (AM) after 8 h IC50 (AM) after 24 h IC50 (AM) after 72 h

1d 37 12 1.5 0.47 0.21

Note: See Materials and Methods section In Vitro Cytotoxicity Assay.

level, as shown by the constant level of both total Rb and pounds from this family are shown to inhibit CDK2 actin. Treatment of HCT 116 cells with compound 1d potently by competing with ATP for binding to the catalytic resulted in induction of caspase-3 activity at 48 and 72 h in subunit of the protein. Co-crystal structures of CDK2 a concentration and time-dependent manner (Fig. 5B). Flow in complex with 1a and 1b reveal that the aminoimi- cytometric analysis of cells treated with 1d resulted in an dazo[1,2-a]pyridine binds in the ATP binding pocket. accumulation of cells in the G2-M phase; however, longer Chemical modification of the substituents at the 3- and 6- exposure to compound (48 and 72 h) resulted in cell death position greatly impacts the potency and selectivity. (Fig. 5C) consistent with the increase in caspase-3 activity. Selectivity for individual CDKs, in this case CDK2, could Similar results were also seen with NCI-H460 (data not be achieved using structural information from CDK2/ shown). These results are consistent with the observed G2-M imidazopyridines co-crystal structures. A remarkable arrest and induction of apoptosis demonstrated by the breakthrough in selectivity with respect to GSK3h was CDK2 specific peptide inhibitor (22). Compound 1d is able demonstrated by imidazopyridine 1d. This compound also to penetrate the cell membrane, inhibit CDK2 activity, selectively inhibits the CDK2-dependent phosphorylation arrest cells in the G2-M phase, and induce apoptosis which of Rb in HCT 116 tumor cells. The discovery of this new appears to contrast with the recent report by Tetsu and class of ATP-site-directed protein kinase inhibitors, amino- McCormick (56). imidazo[1,2-a]pyridines, provides a new medicinal chem- In summary, a new structural class of protein serine/ istry tool in the search for an effective treatment of cancer threonine kinase inhibitors, can be rationally designed and other diseases that involve protein kinase signaling based on the aminoimidazo[1,2-a]pyridine core. Com- pathways.

Figure 5. Imidazopyridine 1d induces cell cycle arrest and apoptosis in HCT 116 cells. A, Western blot analysis of inhibition of CDK2 by imidazopyridine 1d in HCT 116 tumor cells. HCT 116 cells were grown and compound was added for 24 h. Subsequently, cells were lysed and Western blot analysis was performed using the antibodies shown. In vitro, the phosphorylation of T356 of Rb is performed selectively by CDK2, whereas the phosphorylation of S780 is performed selectively by CDK4. B, caspase-3 induction by compound 1d. C, cell cycle effect of imidazopyridine 1d on HCT 116 after 24, 48, and 72 h. Cell cycle analysis was performed as described by Ormerod 57. Compound 1d produces a significant G2- M arrest at 24 h followed by apoptosis at later times.

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Chafiq Hamdouchi, Heather Keyser, Elizabeth Collins, et al.

Mol Cancer Ther 2004;3:1-9.

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