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Identification of N-(4-Piperidinyl)-肝癌-CDK2

Identi?cation of N-(4-Piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxamide (AT7519),a Novel Cyclin Dependent Kinase Inhibitor Using Fragment-Based X-Ray Crystallography and Structure Based Drug Design?

Paul G.Wyatt,?,?Andrew J.Woodhead,?,*Valerio Berdini,⊥John A.Boulstridge,?Maria G.Carr,?David M.Cross,# Deborah J.Davis,|Lindsay A.Devine,§Theresa R.Early,?Ruth E.Feltell,|E.Jonathan Lewis,#Rachel L.McMenamin,| Eva F.Navarro,?Michael A.O’Brien,?Marc O’Reilly,§Matthias Reule,#Gordon Saxty,?Lisa C.A.Seavers,|

Donna-Michelle Smith,#Matt S.Squires,|Gary Trewartha,?Margaret T.Walker,?and Alison J.-A.Woolford?

Astex Therapeutics Ltd,436Cambridge Science Park,Milton Road,Cambridge,CB40QA,United Kingdom

Recei V ed April3,2008

The application of fragment-based screening techniques to cyclin dependent kinase2(CDK2)identi?ed multiple(>30)ef?cient,synthetically tractable small molecule hits for further optimization.Structure-based design approaches led to the identi?cation of multiple lead series,which retained the key interactions of the initial binding fragments and additionally explored other areas of the ATP binding site.The majority of this paper details the structure-guided optimization of indazole(6)using information gained from multiple ligand-CDK2cocrystal structures.Identi?cation of key binding features for this class of compounds resulted in a series of molecules with low nM af?nity for CDK2.Optimisation of cellular activity and characterization of pharmacokinetic properties led to the identi?cation of33(AT7519),which is currently being evaluated in clinical trials for the treatment of human cancers.

Introduction

Background to Fragment-Based Drug Discovery.Astex has previously described the use of fragment-based X-ray crystallographic screening to identify low-af?nity fragment hits for a range of targets,1,2and the area in general has been reviewed extensively over recent years.3–7Fragment-based screening approaches have become widely used throughout the pharmaceutical industry and can now be regarded as a compli-mentary approach to high-throughput screening.Fragments are low-molecular-weight compounds3(typically100-250Da)with generally low binding af?nities(>100μM)and,as a result, very sensitive biophysical screening methods are frequently used to detect them,such as X-ray crystallography,1,8nuclear magnetic resonance spectroscopy(NMR),9and surface plasmon resonance(SPR).10Fragment screening has a number of advantages over conventional screening methodologies.First, only small libraries of compounds are needed for screening purposes(～200-2000),due to the much greater probability of complimentarity between each fragment and the target than is expected for larger,drug-like compounds.11Second,despite their often very low af?nity,fragments generally possess good ligand ef?ciency(LE a)12and as such form a small number of very high quality interactions.It is possible to optimize fragments to relatively low molecular weight leads with good drug-like properties,and this can be achieved with a limited number of molecules,particularly if good structural data is available.LE is the ratio of free binding af?nity to molecular size,depicted mathematically as LE)-?G/HAC≈-RT ln(IC50)/HAC, where the HAC(heavy atom count)includes all non-hydrogen atoms.This concept can be used to compare hits of widely differing structures and activities and is also a simple way of determining if the optimization of a hit into a lead has been carried out ef?ciently.

Inhibition of Cyclin Dependent Kinases.The cyclin-dependent kinases(CDKs)are a family of serine-threonine protein kinases,which are key regulatory elements in cell cycle progression.The activity of CDKs is critically dependent on the presence of their regulatory partners(cyclins),whose levels of expression are tightly controlled throughout the different phases of the cell cycle.13–15Loss of cell cycle control resulting in aberrant cellular proliferation is one of the key characteristics of cancer,16and it is anticipated that inhibition of CDKs may provide an effective method for controlling tumor growth and hence an effective weapon in cancer chemotherapy.17,18 CDK2/cyclin E,CDK4/cyclin D and CDK6/cyclin D prima-rily regulate progression from the G1(Gap1)phase to the S phase(DNA synthesis)of the cell cycle through phosphorylation of the retinoblastoma protein(Rb).19,20Subsequent progression through S phase and entry into G2(Gap2)is thought to require the CDK2/cyclin A https://www.wendangku.net/doc/0117648311.html,plexes of CDK1and the A or B type cyclins regulate both the G2to M phase transition

?Coordinates of the CDK2complexes with compounds6,7,8,10,11, 12,14,15,18,22,23,28,29,and33have been deposited in the Protein Data Bank under accession codes2VTA,2VTH,2VTM,2VTJ,2VTR, 2VTS,2VTI,2VTL,2VTN,2VTO,2VTP,2VTQ,2VTT,and2VU3, together with the corresponding structure factor?les.

*To whom correspondence should be addressed.Phone:+44(0)1223 226287.Fax:+44(0)1223226201.E-mail:a.woodhead@astex-therapeutics. com.

?Medicinal Chemistry.

§Structural Biology.

|Biology.

⊥Computational Chemistry and Informatics.

#DMPK.

?Current address:Drug Discovery Unit,Division of Biological Chem-istry&Drug Discovery College of Life Sciences,James Black Centre, University of Dundee,Dow Street,Dundee,DD15EH,United Kingdom. Phone:+44(0)1382386231.E-mail:p.g.wyatt@https://www.wendangku.net/doc/0117648311.html,.

a Abbreviations:CDK,cyclin dependent kinase;DCM,dichloromethane; DFG,region of conserved amino acids,aspartic acid,phenyl alanine,glycine; EDC,1-(3-dimethyaminopropyl)-3-ethylcarbodiimide;HP CD,hydrox-ypropyl- -cyclodextrin;HOBT,1-hydroxybenzotriazole hydrate;HOAt, 1-hydroxy-7-azabenzotriazole;LE,ligand ef?ciency;NMP,1-methyl-2-pyrrolidinone;NPM,nucleophosmin;Rb,retinoblastoma protein;T/C,mean tumor volume of treated animals divided by the mean control tumor volume.

J.Med.Chem.2008,51,4986–4999

4986

10.1021/jm800382h CCC:$40.75 2008American Chemical Society

Published on Web07/26/2008

and mitosis.15,17However,not all members of the CDK family are involved exclusively in cell cycle control;CDK2/cyclin E plays a role in the p53mediated DNA damage response pathway and also in gene regulation.21–24CDKs 7,8,and 9are implicated in the regulation of transcription,and CDK5plays a role in neuronal and secretory cell function.25–27

Thus inhibiting CDK enzyme activity may affect cell growth and survival via several different mechanisms and therefore represents an attractive target for therapeutics designed to arrest,or recover control of,the cell cycle in aberrantly dividing cells.Accumulating evidence from genetic knockouts of the CDKs and/or their cyclin partners and from siRNA studies suggests signi?cant redundancy in their regulation of key cell cycle events.28–32In addition,the effects of CDK inhibitors on cell proliferation and the induction of apoptosis are not fully reconciled with the current understanding of the biological functions of individual CDKs and the CDK family as a whole.Therefore,an inhibitor active against more than one of the key CDKs may have additional bene?ts in terms of antitumor activity.

Not surprisingly,with the wealth of underlying biological rationale,the development of chemical modulators of CDKs as new anticancer agents has engendered signi?cant interest,with several compounds in clinical and preclinical development.33,34First generation CDK inhibitors such as 1(Flavopiridol/L868275)35and 7-hydroxystaurosporine 36(UCN-01)have been evaluated in the clinic for some time,and recently 1has been granted orphan drug status for the treatment of chronic lym-phocytic leukemia.37Inhibitors with greater selectivity for the CDKs such as 2(roscovitine/CYC-202),383(BMS-387032/SNS-032)39(both primarily target CDK2,but also possess signi?cant CDK7and 9activity),and 4(PD0332991)40(a selective CDK4/6inhibitor)(Figure 1)are currently being evaluated in phase I and II clinical trials,but limited results have been published to date.

Hit Identi?cation.Apo crystals of CDK2were soaked with cocktails of targeted fragments (4fragments per cocktail).The screening set of about 500compounds was made up from a focused kinase set,a drug fragment set,and compounds identi?ed by virtual screening against the crystal structure of CDK2.1Multiple (>30)low-af?nity fragment hits were identi-?ed that bind in the adenosine 5′-triphosphate (ATP)binding

site.A conserved structural feature of all the bound fragments was one or more hydrogen bonding interactions to key backbone residues at the hinge region of CDK2(Glu81and Leu83).ATP itself adopts a similar binding mode,as illustrated in Figure 2.41The compounds shown in Figure 3are a representative selection of the hits identi?ed during fragment screening (compounds 5-8).The hits had only low potency (40μM to 1mM)but were highly ef?cient binders given their low molecular weight (<225)and limited functionality.An important consid-eration during our fragments-to-leads phase is pursuing multiple series in parallel in order to have two or more series for optimization in the later stages of the project.A key feature of this process was the collection of multiple protein -ligand crystal structures to guide iterative cycles of optimization.1,2

To enable the design process,a detailed analysis of the ATP binding site of CDK2and binding mode of known CDK inhibitors was carried out.The analysis identi?ed a number of key interactions and regions of the protein to target in order to optimize activity and physicochemical properties (Figure 2).Hydrogen bonds to the backbone carbonyl and NH of Leu83and the backbone carbonyl of Glu81were commonly observed with bound fragments and more potent ligands.Making all three of these interactions with the hinge appeared to be a potential way to design a very potent and ligand ef?cient inhibitor.Other key areas to explore included the relatively small region between the gatekeeper residue (Phe80)and the catalytic aspartic acid (Asp145)of the DFG motif and a hydrophobic pocket leading to the solvent exposed region (de?ned by Phe82,Ile10,Leu134,and side chain methylene of Asp86).A number of inhibitors in the literature appeared to interact with Asp86,and this was targeted with some early compounds.42,43The solvent accessible region toward Lys89was identi?ed as potentially suitable for modulating physicochemical properties,particularly for

increas-

Figure 1.CDK inhibitors currently under investigation in clinical

trials.

Figure 2.X-ray crystal structure of ATP (adenosine triphosphate)bound into the active site of CDK2(1HCL).The ligand is anchored in place with two hydrogen bonds,one between the backbone carbonyl of Glu81and the 6-amino group and one between the backbone NH of Leu83and the N1position of the adenine ring.An additional favorable electrostatic interaction is made between the hydrogen atom at the C2position and the carbonyl of Leu83.The ribose and phosphate groups form multiple polar interactions,one of which involves coordination to the catalytic magnesium (gray/silver sphere)via the phosphate groups along with Asp145and Asn132.Other key features to notice are that ATP does not interact with the solvent accessible region or the hydrophobic pocket between the gatekeeper residue and the DFG region.

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Figure 3.Fragment -protein cocomplexes of four low-molecular-weight hits identi?ed by fragment-based X-ray crystallographic screening (5-8).On the left is shown the fragment structure and available IC 50data,in the center a pictorial representation of the protein -ligand complex,and the right-hand column provides a description of the experimentally determined binding mode.Key:red spheres,water molecules;purple dashed lines,protein -ligand and water -ligand hydrogen bonds;blue dashed lines,other electrostatic interactions.The PDB code for compound 5is 1WCC.

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Figure 4.Fragment to lead optimization of pyrazine-based https://www.wendangku.net/doc/0117648311.html,pound 5possesses reasonable growth points toward the gatekeeper residue (Phe80)and from the amino group out toward the solvent exposed region.Hydrophobic space ?lling by substitution at the 2-amino position with an aryl group gave compound 9(7μM;LE )0.50),displaying a 150-fold jump in activity over the starting fragment 5.Perhaps surprisingly,the structural data quality for this compound was poor,with no electron density observed for the aryl group,possibly due to the aryl group being able to bind in a number of conformations.Introduction of a sulfonamide at the 4-position of the aryl group forces an intramolecular salt bridge between Asp86and Lys89to break and allows for the formation of a further H-bonding interaction between the sulfonamide and the backbone NH of Asp86.In spite of this additional interaction,only a modest increase in activity is observed for 10(1.9μM;LE )0.43).51Further modi?cation of this group or replacement of the 6-chloro substituent suggested that optimization beyond low micromolar activity was not straightforward,so this series was not pursued further.Key:red spheres,water molecules;purple dashed lines,protein -ligand and water-ligand hydrogen bonds;blue dashed lines,other electrostatic

interactions.

Figure 5.Fragment to lead optimization of pyrazolopyrimidine-based inhibitors.Fragment 8binds to CDK2as described in Figure 3.The binding mode is very similar to that of Roscovitine (2)and other bicyclic templates described in the literature.Substitution of compound 8at the 7-position with a hydrogen bond donor allowed a third interaction to be formed with the protein backbone at the hinge region (carbonyl of Leu83).The amine could be substituted with a range of functionalities and the isopropyl group being particularly https://www.wendangku.net/doc/0117648311.html,pound 11gave a 700-fold jump in binding af?nity (IC 50)1.5μM,LE )0.50)and an improvement in ligand ef?ciency over the starting fragment.Growing out from the 5-position allowed the opportunity to access the ribose and phosphate binding regions of the active site.Introduction of basic functionality in the phosphate binding pocket was well tolerated,with this strategy producing the high af?nity lead 12(IC 50)0.03μM,LE )0.45).The crystal structure shows the 4-amino group to be forming hydrogen bonds with the carboxylate of Asp145and the side chain carbonyl of Asn132,mimicking the Mg 2+observed in many ATP bound kinase https://www.wendangku.net/doc/0117648311.html,pound 12also displayed good cellular activity (HCT116cell IC 50)0.29μM),however,this did not translate into in vivo activity and work on this series was abandoned in favor of more promising compounds.Key:red spheres,water molecules;purple dashed lines,protein -ligand and water -ligand hydrogen bonds;blue dashed lines,other electrostatic interactions.

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ing water solubility.Many of these interactions are discussed in an interesting review by Liao on the molecular recognition of protein kinase binding pockets.44Results and Discussion

Summary of Fragment to Lead Optimization.Figure 3shows four representative fragment hits identi?ed by structural screening of CDK2.A number of considerations were taken into account when deciding which of the hits should be worked on further,and these included LE,vectors suitable to access the key regions highlighted in Figure 2,novelty,and synthetic tractability.Of the fragments described,compound 7was assessed not to have suitable vectors for optimization and,in addition,the chemistry did not appear to be very tractable.As a result,this compound did not enter hit-to-lead https://www.wendangku.net/doc/0117648311.html,pounds 5,6,and 8were deemed to have suitable vectors and the chemistry suf?ciently tractable to warrant further work.The optimization of compounds 5and 8is outlined in brief in Figures 4and 5,respectively;the optimization of compound 6will be discussed in detail in the following sections.

Fragment to Lead Optimization of Compound 6.The “hit to lead”chemistry of 6focused primarily on two vectors (from the 3and 5positions of the indazole ring,see Figure 6)suitable to access pockets identi?ed by the kinase structural analysis (Figure 2).Structural data from Astex fragments and known CDK inhibitors suggested that formation of an additional hydrogen bonding interaction to the carbonyl of Leu83was a possibility.45,46This was achieved by linking an aryl amide to the 3-position of indazole,resulting in 13(IC 50)3μM;LE )0.42)(Table 1).The phenyl ring was twisted out of plane and occupies the hydrophobic pocket formed by the backbone of the linker region and side chains of Ile10and Leu134.Addition of a sulfonamide group at the 4-position of the phenyl ring afforded 14with submicromolar activity (IC 50)0.66μM;LE )0.38).The sulfonamide picks up two further interactions,both to Asp86,a direct hydrogen bond to the backbone NH and a water-mediated interaction to the carboxylate side chain (Figure 6).42,43

Substitution of the indazole ring at the 4or 5positions resulted in relatively small increases in CDK2activity,while as expected,substitutions at the 6or 7positions were poorly tolerated (data not shown)due to the close proximity of Phe80.An alternative strategy was pursued in parallel and rather than seeking to increase the potency of 14by continuing to add molecular weight,the system was simpli?ed by removal of the fused benzene ring to afford the pyrazole 15(IC 50)97μM;

LE )0.39).Despite a drop in activity,the LE was similar to the more elaborated compounds and the binding mode

Figure 6.CDK2cocrystal structures of compounds 6,14,and 15.Key:red spheres,water molecules;purple dashed lines,protein -ligand hydrogen bonds;arrows indicate potential vectors for substitution.

Table 1.CDK2Inhibition of Selected Indazole and Pyrazole Amides

Compound were assayed 2or more times.b IC 50data for 2was generated in house and compares well with values quoted in the literature (within 2fold).47

4990Journal of Medicinal Chemistry,2008,Vol.51,No.16Wyatt et al.

con?rmed by the X-ray crystal structure remained identical to the starting fragment,which encouraged us to pursue this strategy further.The change of hinge binder provided a signi?cantly different vector and improved access to the DFG region between the gatekeeper residue (Phe80)and the catalytic aspartate (Asp145)(Figure 2).It appeared that derivatizing the pyrazole at the 4-position presented an opportunity to grow into this pocket (Figure 6).Accordingly,introduction of a 4-amino group as a synthetic handle gave 17which resulted in a modest increase in activity (IC 50)85μM;LE )0.35).Introduction of a hydrogen bond acceptor gave compounds such as the amide 18,leading to a 100-fold increase in activity and improved LE (IC 50)0.85μM;LE )0.44).An X-ray structure of 18bound into CDK2showed that the increase in activity was at least in part due to a water mediated hydrogen bond from the acetamide carbonyl oxygen to the backbone NH of Asp145(Figure 7).Another important observation is that the planarity of 18is achieved due to an intramolecular hydrogen bond between the acetamide NH and the benzamide carbonyl,allowing the compound to ?t into the narrow binding pocket.

Ongoing with this work were attempts to replace the amide at the 3-position of the pyrazole with alternative groups that could maintain the hydrogen bonding interaction to the backbone carbonyl of Leu83while maintaining the planarity of the system.For example,a 2-benzimidazole group proved to be an effective replacement for the aryl amide.16(IC 50)25μM;LE )0.45)is more active than the corresponding amide 15.Further optimization of 16led to the identi?cation of an alternative series with excellent kinase and cell activity.Details of the develop-ment of this alternative series will follow in a subsequent publication.

The protein -ligand structure of 18indicated that the methyl of the acetamide group is in very close proximity to the side chain carboxylate of Asp145(approximately 3.5?),making the pocket relatively small.Some protein ?exibility is observed in this region of the binding site,so in order to probe this area further,a limited number of amides with diverse properties were synthesized (Table 2).A range of simple functionalities such as 19and 20did not afford a signi?cant increase in activity over 18;however,interesting levels of kinase activity were obtained with directly attached monocycles such as the cyclo-hexyl amide 21and benzamide 22,and these compounds also showed some indication of cellular activity.The benzamide 22was particularly interesting,showing only a small increase in binding af?nity and a decrease in ligand ef?ciency (IC 50)0.14

μM,LE )0.39);however,the protein -ligand crystal structure (Figure 7)provided a number of important insights into the binding mode of this compound.First,a small amount of protein movement had occurred,allowing the aromatic ring to be accommodated.Second the phenyl ring was signi?cantly twisted out of plane of the amide,with a torsion angle of 51°,an energetically unfavorable conformation.It was postulated that stabilization of this twist by diortho substitution of the phenyl ring might be bene?cial.The X-ray structure con?rmed that 23bound to CDK2as predicted (Figure 7),resulting in a 45-fold increase in kinase activity for the addition of only two heavy atoms and with a ligand ef?ciency very similar to the starting fragment (IC 50)0.003μM,LE )0.45).

Although 23exhibited good kinase activity and its pharma-cokinetic (PK)properties indicated it to be a good lead

molecule,

Figure 7.CDK2cocrystal structures of compounds 18,22,and 23,demonstrating the use of 2,6-disubstitution on the phenyl ring (23)to stabilize the induced twist observed for the benzamide of 22on binding to CDK2.Key:red spheres,water molecules;purple dashed lines,protein -ligand and water-ligand hydrogen bonds.

Table 2.Pyrazole Diamide Structure -Activity Relationships

(SAR)

Compounds were assayed 2or more times unless indicated *where n )1.

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with moderate plasma clearance (40mL/min/kg)after intrave-nous (iv)dosing in mice,its antiproliferative cell activity against HCT116colon cancer cells was only moderate (1.4μM).One explanation for this moderate cell activity may be due to low cell permeability.23has a ClogP of 2.4,however,the measured value is approximately 2log units higher,presumably due to internal hydrogen bonding reducing the overall polarity of the molecule.This relatively high lipophilicity may be detrimental to cell permeability,and in an attempt to address this,further optimization of the series was sought by replacing the lipophilic 4-?uorophenyl group of 23(Table 3).In general,other aromatic groups (data not shown)and simple alkyl groups such as in 24gave good kinase activity but only moderate cell activity.However,the directly attached cycloalkyl ring of 25gave an improvement in kinase activity and was the ?rst compound in this series with submicromolar cell activity.Because of its reasonable cellular activity the PK properties of 25were determined in mice and it was found to have high plasma clearance (65mL/min/kg).A potential cause of this was oxidative metabolism of the highly lipophilic cyclohexyl group,and in an attempt to address this,modi?cations were made to the cyclohexyl ring.Although a number of 4-substituted cyclohexyl derivatives such as 26and 27exhibited good kinase and cell activity,most had relatively high plasma clearances.Introduction of a nitrogen atom into the ring affording 28and 29gave compounds with good CDK2and cell potency.It became apparent from making these small polar changes that modulating physicochemical properties was just as important as increasing kinase activity when attempting to improve cell potency.Interestingly,the 3-piperidinyl isomer 29exhibited an

increase in CDK1as well as CDK2activity compared to 28.This increase in activity can be explained by the presence of a conserved carboxylate residue in CDK1and CDK2(Asp86in CDK2)with which the 3-piperidinyl nitrogen of 29can interact.The X-ray structure of 29bound to CDK2(Figure 8)con?rms this interaction,the protonated nitrogen being 2.65?from the carboxylate of https://www.wendangku.net/doc/0117648311.html,pound 28was considered to be a promising lead due to its reasonable cell activity and acceptable plasma protein binding (PPB)(Table 5);as a consequence of this,further in vivo characterization of 28was performed.Despite showing moderate plasma clearance (43mL/min/kg),the compound was dosed to mice bearing HCT116tumor xenografts to determine the level of compound in the tumor.After a single 10mg/kg dose of 28was administered via the ip route,reasonable,albeit somewhat variable levels of compound appeared to distribute into tumor (AUC )3422(2478h ·ng/g)(Table 5).The compound was much more rapidly cleared from plasma,with negligible material remaining after 7h (data not shown).In an attempt to determine whether compound levels in tumor were a potential indicator of in vivo ef?cacy,28was evaluated for antitumor activity in a mouse xenograft model.The hydrochloride salt of compound 28was dosed ip bid (twice daily)at 18.2,9.1,and 4.6mg/kg to SCID mice bearing early stage HCT116human colon carcinoma xenografts for https://www.wendangku.net/doc/0117648311.html,pound 28showed a clear dose -response for antitumor

Table 3.Summary of SAR for Compounds 23-

Compounds were assayed 2or more times unless indicated *where n )1.b The plasma clearance data was determined after IV administration to BALB/c mice at 0.2mg/kg according to Pharmacokinetic Study

Methods.

Figure 8.CDK2cocrystal structures of compounds 28and 29.29forms an additional hydrogen bond between the ring nitrogen of the piperidyl group and the carboxylate of Asp86.This may explain the observed improvement in binding af?nity.Key:red spheres,water molecules;purple dashed lines,protein -ligand and water -ligand hydrogen bonds.

4992Journal of Medicinal Chemistry,2008,Vol.51,No.16Wyatt et al.

activity although the dose schedule was not optimized.The 18.2mg/kg dose group showed tumor growth inhibition of 86%(%T /C )14),however this dose was not well tolerated.At a tolerated dose of 9.1mg/kg,growth inhibition was 46%(%T /C )54)(Table 5)and the 4.6mg/kg group showed 22%growth inhibition (%T /C )78).These data suggested that distribution of compounds in tumor and their persistence there might be a useful indicator of in vivo activity when considered alongside protein binding and cell potency.The further impact of compound disposition on biomarker modulation will be dis-cussed in a subsequent paper.

Further optimization of 28aimed at increasing activity against CDK2and subsequently improve cell activity was explored by reoptimizing the 2,6-di?uorophenyl moiety (Table 4).Attempts to block potential metabolism of the phenyl ring by substitution of the 4-position,e.g.,in 30resulted in somewhat reduced inhibitory activity against the kinase.Replacement of one of the ?uorines of 28with small substituents such as methoxy and chloro to give 31and 32was well tolerated and resulted in increased kinase and cell activity (Table 4).The 2,6-dichlo-rophenyl derivative 33gave an increase in kinase and cell activity,as the chlorine atoms ?lled this lipophilic pocket more

effectively than ?uorine.Because previous compounds showed persistence in tumor in spite of moderate to high plasma clearance (e.g.,28),further compounds (31and 33)were dosed to HCT116tumor bearing mice at 10mg/kg to determine tumor distribution properties.A similar trend to compound 28was observed,with signi?cant levels of both 31and 33present in tumor (AUC )3325(543and 6260-6340h ·ng/g,respec-tively)(Table 5).Compound 33in particular showed good tumor exposure.

The promising in vitro kinase and antiproliferative cell activity,coupled with low PPB and reasonable tumor distribu-tion for both 31and 33,led them to be evaluated in vivo for potential antitumor ef?https://www.wendangku.net/doc/0117648311.html,pound 31showed 38%tumor growth inhibition (%T /C )62)in the HCT116mouse xenograft model at 10mg/kg,although the dose and schedule were not https://www.wendangku.net/doc/0117648311.html,pound 33showed signi?cant ef?cacy in the same tumor type producing tumor growth inhibition of 87%(%T /C )13)when dosed at 9.1mg/kg ip bid for 10days (Table 5),which warranted further investigation.A similar bene?cial effect was observed for 33in the A2780(human ovarian carcinoma cell line)mouse xenograft model.Details of this and further characterization of 33is described in the following section.

Characterization of Compound 33.Kinase Selectivity Pro?https://www.wendangku.net/doc/0117648311.html,pound 33was pro?led more widely against a panel of kinases (see Supporting Information).In addition to CDKs 1and 2(IC 50s 190nM and 47nM,respectively),33potently inhibited a number of other CDKs (4and 5in particular,IC 50s 67nM and 18nM,respectively),but had lower activity against other kinases tested (more detailed selectivity data will be published in a subsequent paper).One explanation for the observed selectivity over some kinases (Aurora A,IR kinase,MEK,PDK1,c-abl,IC 50>10μM)is shown in Figure 9a.All these kinases possess an additional glycine residue (in between the amino acids corresponding to Gln85and Asp86of CDK2),which causes the main chain to bulge into the ATP binding pocket resulting in a clash with the piperidine of 33.

Cell-Based https://www.wendangku.net/doc/0117648311.html,pound 33is a potent inhibitor of HCT116cell proliferation (used as a primary screen during lead optimization).Following 72h exposure,33potently inhibited the proliferation of a range of human tumor cell lines (over 100cell lines have been tested),with compound 33showing sub 1μM activity against more than 75(data not shown).Compound 33had reduced antiproliferative activity against the nontrans-formed ?broblast cell line,MRC-5,but more signi?cantly,it did not affect the viability of noncycling MRC-5cells at doses up to 10μM (Table 6).These data suggest that the antiprolif-erative activity is cell cycle related and not due to general cytotoxicity to nondividing cells.

The mechanism of action of 33in cells was investigated by monitoring the phosphorylation state of substrates speci?c for the various CDKs,following treatment with 33for 24h.These studies indicated that inhibition of phosphorylation of the CDK1substrate PP1R (Thr320)and the CDK2substrates Rb (Thr821)and Nucleophosmin (NPM)(Thr199)(data to be published in

Table 4.Substituted Benzamide SAR for Compounds 30-

Compounds were assayed 2or more times.b The plasma clearance data was determined after IV administration to BALB/c mice at 0.2mg/kg according to Pharmacokinetic Study Methods.

Table 5.Activity,Distribution,and Initial Ef?cacy Parameters for Compounds 28,31,and 33Compound

HCT116a IC 50(μM)Plasma protein binding

(%bound)

Tumor AUC (0-t )h ·ng/g b

Ef?cacy screening protocol in HCT116tumor xenograft model %T /C e

280.31843422(2478(n )4)9.1mg/kg/dose bid QDx10c 54310.052753325(543(n )3)10mg/kg/dose bid QDx9d 6233

0.082

6260-6340

9.1mg/kg/dose bid QDx10c

Compounds were assayed 2or more times.b Mean (SD for n determinations or range for n )2determinations.c Ef?cacy study conducted in SCID mice following ip administration.d Ef?cacy study conducted in BALB/c nude mice following ip administration.e %T /C is the mean tumor volume of treated animals divided by the mean control tumor volume expressed as a percentage.

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a subsequent paper)in HCT116cells occurred at doses consistent with the observed antiproliferative effects.

Pharmacokinetics Study.As summarized in Table 7,the systemic clearance of 33in BALB/c mice after iv dosing averaged 46mL/min/kg with a mean half-life (t 1/2)and volume of distribution (V ss )of 0.68h and 1.6L/kg,respectively.33showed low oral bioavailability (<1%),which was a common feature of many closely related basic compounds.

In Vivo Antitumor https://www.wendangku.net/doc/0117648311.html,pound 33was evaluated for its in vivo antitumor activity in nude BALB/c mice bearing early stage A2780human ovarian carcinoma xenografts with a mean starting volume of approximately 50mm 3(Figure 10).In this study,the hydrochloride salt of 33,dissolved in 0.9%saline,was administered by the intraperitoneal (ip)route,twice daily,for 8consecutive days.Tumor growth inhibition at the end of the experiment was 86%at the 7.5mg/kg dose level (%T /C )14).A more extensive biological characterization of compound 33,including a comprehensive cell cycle analysis and a detailed in vivo ef?cacy evaluation will be published separately.

https://www.wendangku.net/doc/0117648311.html,pounds 13,14,and 15were synthesized by coupling either 1H -indazole-3-carboxylic acid or 1H -pyrazole-3-carboxylic acid with aniline or https://www.wendangku.net/doc/0117648311.html,pound 16was prepared by coupling 1H -pyrazole-3-carboxylic acid with 1,2-diaminobenzene followed by acid mediated cyclization to form the benzimidazole.Synthesis of aminopyrazole 17was achieved (Scheme 1)by coupling

4-nitropyrazole-3-carboxylic acid 34with 4-?uoroaniline,fol-lowed by catalytic hydrogenation of 35with palladium on carbon.Diamides 18-23were synthesized by coupling of 17with acetic anhydride or the appropriate carboxylic acids using EDC/HOBt.

Esteri?cation of 34with ethanol under acidic conditions followed by catalytic hydrogenation of the nitro group gave the aminopyrazole 36(Scheme 2).Acylation of 36with 2,6-di?uorobenzoic acid,followed by basic hydrolysis afforded pyrazole acid 37.Coupling of 37with the required amines using EDC/HOBt gave compounds https://www.wendangku.net/doc/0117648311.html,pounds 27-29required an additional deprotection step to remove the N-tert -butoxycarbonyl group.This was achieved under standard acidic conditions with saturated HCl in EtOAc.The piperidine amides 30-33were synthesized via the aminopyrazole 38(Scheme 3),itself synthesized from the coupling of 4-amino-piperidine-1-carboxylic acid tert -butyl ester and 34,followed by reduction of the nitro group.Coupling of 38with the

appropriate

Figure 9.(a)Overlay of C-R traces of the crystal structure of 33bound into CDK2(green)with the crystal structures of Aurora A (PDB code 1OL6,yellow),insulin receptor (IR)kinase (1GAG,brown),MEK1(1S9J,purple),PDK1(1UU3,blue),and c-abl (1IEP,red),showing the clash of the kinases (except CDK2)with the piperidine of 33.Aurora A,IR kinase,MEK1,PDK1,and c-abl all have IC 50values >10μM.(b)CDK2cocrystal structures of compound 33.Key:red spheres,water molecules;purple dashed lines,protein -ligand and water -ligand hydrogen bonds.(c)Molecular surface representation of CDK2with 33bound in the ATP binding site.The piperidine moiety is pointing out of the pocket toward solvent.The twisted 2,6-dichlorophenyl is toward the back of the pocket,with the two chlorine atoms ef?ciently ?lling small hydrophobic pockets.Table 6.In Vitro Antiproliferative Activity of 33in a Panel of Human Tumor Cell Lines

Origin Cell line

IC 50(nM)colon carcinoma HCT11682ovarian carcinoma A2780350?broblast

MRC 5

980MRC 5(nonproliferative)

>10000

Table 7.Mouse PK Data of Compound 33after iv Administration to BALB/c Mice a

t 1/2(h)Cl (ml/min/kg)

V ss (L/kg)0.68(0.28

46(21

1.6(0.9

Mean (SD for n )5

-7replicate studies.

Figure 10.Ef?cacy of 33in the early stage A2780ovarian carcinoma xenograft mouse model following repeated administration at 7.5mg/kg/dose given twice daily by the intraperitoneal route for 8days against a matched vehicle-dosed control.Tumor growth curves (SE for groups of n )12.Growth curves became signi?cantly different from control from day 5onward (*p <0.05,**p <0.001).The CDK2and cellular IC 50data quoted is the mean based on a minimum of two separate determinations.

4994Journal of Medicinal Chemistry,2008,Vol.51,No.16Wyatt et al.

carboxylic acids,then acidic deprotection of the Boc protected piperidine of 39,afforded compounds 30-33.During preclinical development,compound 33was synthesized by a six-step route (similar to Scheme 2)in multikilogram quantities with an overall yield of 80%and purity >99%,without the requirement for puri?cation by column chromatography.Conclusions

High-throughput X-ray crystallographic screening of fragment libraries was used to identify multiple hits that bound to CDK2.A number of these fragment hits were elaborated in parallel with the aid of detailed structural information enabling the project to take 3series into late stage lead optimization.Optimization of the indazole hit 6eventually led to the discovery of AT7519(33).The key compounds in the discovery of 33are summarized in Scheme 4.Important structural features identi?ed throughout the optimization process include (Figure 9b):(i)a donor -acceptor -donor interaction anchoring the molecule to the hinge region of CDK2(residues Glu81and Leu83),(ii)a water mediated hydrogen bond between the carbonyl of the 4-benzamide group and the backbone N -H of Asp145,(iii)stabilization of the twisted benzamide conformation by introduction of two ortho-substituents,(iv)introduction of the solubilizing aminopiperidine amide group resulted in improved hydrophobic ?lling of the region bounded by the backbone of the hinge and sidechains of residues Phe82,Ile10,Leu134,and Asp86and which led to selectivity over non-CDK kinases,improved cellular activity,and lower plasma clearance.During the fragment to lead process,structural data and LE were

used to ensure optimization was carried out ef?ciently.It quickly became apparent for indazole based compounds (e.g.,14)that molecular weight was being added for only small gains in potency.In contrast,introduction of the acetamide moiety to the pyrazole system (18)and stabilizing the twisted benzamide conformation (23)were very ef?cient methods of increasing enzyme activity.During later stage lead optimization,LE became a less important criterion as multiple parameters were being changed simultaneously.28is a good example of a compound showing a small decrease in enzyme activity,however,this is offset by a change in physicochemical properties leading to an improvement in cellular activity.

Compound 33is a potent,ligand ef?cient inhibitor of CDK2(IC 50)0.047μM;LE )0.42),with good activity against a range of human tumor cell lines.It has a good pro?le against the major cytochrome P450isoforms (<30%inhibition at 10μM for 1A2,2D6,3A4,2C9,2C19),has good aqueous thermodynamic solubility either as the acetate or hydrochloride salt (>25mg/ml in water or 0.9%saline),and its synthetic tractability makes it readily amenable to large scale synthesis.On the basis of these and further data (to be published in a later paper),compound 33was selected as a preclinical development candidate and subsequently entered clinical development.Experimental Section

Chemistry.Reagents and solvents were obtained from com-mercial suppliers and used without further puri?https://www.wendangku.net/doc/0117648311.html,pounds 5-8were obtained from commercial suppliers.Thin layer chro-

Scheme 1

Reagents and conditions:(a)4-?uoroaniline,EDC,HOBt,DMF;(b)10%Pd/C,EtOH,H 2;(c)Ac 2O,pyridine,or RCO 2H,EDC,HOBt,DMF,or DMSO.

Scheme 2

Reagents and conditions:(a)SOCl 2,EtOH;(b)10%Pd/C,EtOH,H 2;(c)2,6-di?uorobenzoic acid,EDC,HOBt,DMF;(d)NaOH,MeOH/H 2O (1:1);(e)RNH 2,EDC,HOBt,or HOAt,DMF,or DMSO;(f)saturated solution of HCl in EtOAc (where R contains a Boc protected amine).

Scheme 3

Reagents and conditions:(a)4-amino-piperidine-1-carboxylic acid tert-butyl ester,EDC,HOBt,DMF;(b)10%Pd/C,EtOH,H 2;(c)RCO 2H,EDC,DMF;(d)saturated solution of HCl in EtOAc.

Identi?cation of AT7519Journal of Medicinal Chemistry,2008,Vol.51,No.164995

matography (TLC)analytical separations were conducted with E.Merck silica gel F-254plates of 0.25mm thickness and were visualized with UV light (254nM)and/or stained with iodine,potassium permanganate,or phosphomolybdic acid solutions fol-lowed by heating.Standard silica gel chromatography was employed as a method of puri?cation using the indicated solvent https://www.wendangku.net/doc/0117648311.html,pound puri?cation was also carried out using a Biotage SP4system using prepacked disposable SiO 2cartridges (4,8,19,38,40,and 90g sizes)with stepped or gradient elution at 5-40mL/min of the indicated solvent mixture.Proton nuclear magnetic resonance (1H NMR)spectra were recorded in the deuterated solvents speci?ed on a Bruker Avance 400spectrometer operating at 400MHz.Chemical shifts are reported in parts per million (δ)from the tetramethylsilane internal standard.Data are reported as follows:chemical shift,multiplicity (br )broad,s )singlet,d )doublet,t )triplet,m )multiplet),coupling constants (Hz),https://www.wendangku.net/doc/0117648311.html,pound purity and mass spectra were determined by a Waters Fractionlynx/Micromass ZQ LC/MS platform using the positive electrospray ionization technique (+ES),or an Agilent 1200SL-6140LC/MS system using positive -negative switching,both using a mobile phase of acetonitrile/water with 0.1%formic acid (see Supporting Information for experimental details).

4-(6-Chloropyrazin-2-ylamino)benzenesulphonamide (10).A suspension of 2,6-dichloropyrazine (100mg,0.67mmol)and 4-sulfamoyl aniline (115mg,0.67mmol)in NMP in a sealed tube was heated to 250°C under microwave irradiation for 8min.The mixture was diluted with water and extracted with EtOAc.The organic layer was ?ltered and evaporated under reduced pressure.The residue was puri?ed by preparative HPLC (method C)to give 10(11mg,6%).1H NMR (400MHz,DMSO-d 6):δ10.23(s,1H),8.26(s,1H),8.11(s,1H),7.79(s,4H),7.23(s,2H).LC/MS:R t )1.08min,[M +H]+285(method A).

5-Chloro-7-isopropylaminopyrazolo[1,5-a ]pyrimidine-3-carbo-nitrile (11).Isopropylamine (0.12mL,2.83mmol)and potassium carbonate (652mg,4.72mmol)were added to a solution of 5,7-dichloropyrazolo[1,5-a ]pyrimidine-3-carbonitrile (500mg, 2.36mmol)in acetonitrile (10mL).The resulting mixture was stirred at room temperature overnight,?ltered,and concentrated in vacuo to give 11(10mg,2%).1H NMR (400MHz,Me-d 3-OD):δ8.39(s,1H),6.50(s,1H),4.09-3.98(m,1H),1.39(d,J )6.4Hz,6H).LC/MS:R t )2.81min,[M +H]+236(method A).

5-(4-trans-Amino-cyclohexylamino)-7-isopropylamino-pyrazo-lo[1,5-a ]pyrimidine-3-carbonitrile formate (12).A solution of 5-chloro-7-isopropylamino-pyrazolo[1,5-a ]pyrimidine-3-carboni-trile (180mg,0.61mmol),trans -1,4-cyclohexanediamine (84mg 0.73mmol)and N ,N -diisopropylethylamine (160μL)in DMF (3.1mL)was subjected to microwave heating at 140°C for 1h.The reaction mixture was allowed to cool and then puri?ed by RP-HPLC (method C)to afford 12as a colorless solid (35mg,18%).1

H NMR (400MHz,DMSO-d 6):δ8.42(s,1H),8.23(s,1H),7.27-7.10(m,2H),5.41(s,1H),3.80(s,1H),3.55(s,2H),2.89(t,J )12.3Hz,1H),1.96(t,4H),1.46-1.32(m,2H),1.32-1.22(m,8H).LC/MS:R t )1.03min (RR),[M +H]+314(method B).

Scheme 4.Key Compounds in the Optimization of Fragment 6to the Clinical Candidate 33

IC 50data are for CDK2.

4996Journal of Medicinal Chemistry,2008,Vol.51,No.16Wyatt et al.

1H-Indazole-3-carboxylic Acid(4-Sulfamoylphenyl)amide(14). To1H-indazole-3-carboxylic acid(100mg,0.6mmol)in DMF(5

mL)was added EDC(142mg,0.72mmol),HOBt(100mg,0.72

mmol),and sulfanilimide(127mg,0.72mmol).The reaction was

heated at100°C for48h then cooled and evaporated.The residue

was partitioned between DCM and saturated NaHCO3solution,then

the resultant solid was collected by?ltration,washed with water

and DCM,and then dried.The crude material was puri?ed by RP-

HPLC(method C),and product containing fractions were combined

and evaporated to give14as a white solid(12mg,6%).1H NMR

(400MHz,DMSO-d6):δ13.87(s,1H),10.68(s,1H),8.24(d,J )8.1Hz,1H),8.09(d,J)8.3Hz,2H),7.80(d,J)8.3Hz,2H), 7.69(d,J)8.4Hz,1H),7.48(t,J)7.6Hz,1H),7.32(t,J)7.7

Hz,1H),7.26(s,2H).LC/MS:R t)2.72[M+H]+317(method

A).

1H-Pyrazole-3-carboxylic Acid Phenylamide(15).1H-Pyrazole-3-carboxylic acid(100mg,0.9mmol),EDC(211mg,1.1mmol), HOBt(147mg,1.1mmol),and aniline(0.089mL,0.98mmol) were dissolved in DMF(5mL)and the resulting solution was stirred for2days.The mixture was concentrated under reduced pressure and partitioned between water and EtOAc.The organic fraction was dried(MgSO4),?ltered,and evaporated in vacuo and the residue puri?ed by column chromatography to give15(97mg, 58%)as a white solid.1H NMR(400MHz,DMSO-d6):δ13.44 (s,1H),10.01(s,1H),7.93-7.84(s,1H),7.81(d,J)8.0Hz, 2H),7.33(dd,8.0,7.7Hz,2H),7.08(t,J)7.7Hz,1H),6.82(s, 1H).LC/MS:R t)0.98(RR)[M-H]-186(method B).

4-Nitro-1H-pyrazole-3-carboxylic Acid(4-Fluoro-phenyl)-amide (35).4-Nitro-1H-pyrazole-3-carboxylic acid34(10g;63.66mmol) was added to a stirred solution of4-?uoroaniline(6.7mL;70 mmol),EDC(14.6g;76.4mmol),and HOBt(10.3g;76.4mmol) in DMF(25mL)and then stirred at room temperature for16h. The solvent was removed by evaporation under reduced pressure and the residue triturated with EtOAc/saturated brine solution.The resultant yellow solid was collected by?ltration,washed with2M hydrochloric acid,and then dried under vacuum to give35(15.5 g,97%).1H NMR(400MHz,DMSO-d6):δ14.25(s,1H),10.73 (s,1H),8.96(s,1H),7.72(dd,J)8.4,4.9Hz,2H),7.22(t,J) 8.5Hz,2H).LC/MS:R t)2.92min,[M+H]+250(method A). 4-Amino-1H-pyrazole-3-carboxylic Acid(4-Fluoro-phenyl)-amide(17).Compound35(15g,60mmol)was dissolved in200 mL of EtOH,treated with1.5g of10%palladium on carbon under a nitrogen atmosphere,then hydrogenated at room temperature and pressure for16h.The catalyst was removed by?ltration through celite and the?ltrate evaporated.The crude product was dissolved in acetone/water(200mL,1:1),and after slow evaporation of the acetone,17was collected by?ltration as a brown crystalline solid

(8.1g,62%).1H NMR(400MHz,DMSO-d6):δ12.77(s,1H),

9.87(s,1H),7.88-7.76(m,2H),7.25-7.07(m,3H),4.69(s,2H).

LC/MS:R t)1.58min,[M+H]+221(method A).

4-Acetylamino-1H-pyrazole-3-carboxylic Acid(4-Fluoro-phen-yl)-amide(18).Compound17(500mg;2.27mmol)was dissolved in pyridine(5mL),treated with acetic anhydride(240μL,2.5 mmol),and then stirred at room temperature for16h.The solvent was removed by evaporation,and then dichloromethane(20mL) and2M hydrochloric acid(20mL)were added.The undissolved solid was collected by?ltration,washed with dichloromethane and water,and then dried under vacuum.The product18was isolated as an off-white solid(275mg,46%).1H NMR(400MHz,DMSO-d6):δ13.33(br s,1H),10.29(s,1H),9.54(s,1H),8.24(s,1H), 7.90-7.77(m,2H),7.18(t,J)8.7Hz,2H),2.12(s,3H).LC/ MS:R t)2.96min,[M+H]+263(method A).

4-(2-Hydroxyacetylamino)-1H-pyrazole-3-carboxylic Acid(4-?uorophenyl)amide(19).Hydroxyacetic acid(19mg,0.25mmol) was added to a solution of17(50mg,0.23mmol),EDC(53mg, 0.27mmol),and HOBt(37mg,0.27mmol)in DMF(5mL).The reaction mixture was stirred at room temperature for24h.The solvent was removed under reduced pressure.The residue was puri?ed by preparative LC/MS(method C)and,after evaporation of product-containing fractions,yielded19as a white solid(26mg, 41%).1H NMR(400MHz,DMSO-d6):δ13.45(s,1H),10.42(s,1H),10.37(s,1H),8.38-8.26(m,1H),7.92-7.79(m,2H), 7.25-7.12(m,2H),6.09(t,J)5.6Hz,1H),4.01(d,J)5.6Hz, 2H).LC/MS:R t)2.65min,[M+H]+278(method A).

The following compounds were synthesized using the same method as used for19:

4-Benzoylamino-1H-pyrazole-3-carboxylic Acid(4-Fluoro-phen-yl)-amide(22).Benzoic acid gave22as a pink solid(26mg,35%). 1H NMR(400MHz,DMSO-d6):δ13.55(br s,1H),10.56(s,1H),

10.50(s,1H),8.42(s,1H),7.96-7.81(m,4H),7.70-7.55(m, 3H),7.27-7.14(m,2H).LC/MS:R t)3.96min,[M+H]+324 (method A).

4-(2,6-Di?uoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid (4-Fluoro-phenyl)-amide(23).2,6-Di?uorobenzoic acid in DMSO gave23as a cream-colored solid(25mg,30%).1H NMR(400 MHz,DMSO-d6):δ13.57(s,1H),10.42(s,1H),10.31(s,1H), 8.43(s,1H),7.88-7.77(m,2H),7.69-7.58(m,1H),7.27(dd,J

)8.6,8.5Hz,2H),7.17(dd,J)8.7,8.6Hz,2H).LC/MS:R

t 3.76min,[M+H]+361(method A).

4-Amino-1H-pyrazole-3-carboxylic Acid Ethyl Ester(36).Step 1.Thionyl chloride(2.9mL,39.8mmol)was slowly added to a mixture of34(5.68g,36.2mmol)in EtOH(100mL)at room temperature then stirred for48h.The mixture was evaporated in vacuo and re-evaporated with toluene to afford4-nitro-1H-pyrazole-3-carboxylic acid ethyl ester as a white solid(6.42g,96%).1H NMR(400MHz,DMSO-d6):δ14.4(s,1H),9.0(s,1H),4.4(q, 2H),1.3(t,3H).

Step2.A mixture of4-nitro-1H-pyrazole-3-carboxylic acid ethyl ester(6.40g,34.6mmol)and10%Pd/C(650mg)in EtOH(150 mL)was stirred under an atmosphere of hydrogen for20h.The mixture was?ltered through a plug of celite,evaporated under vacuum then re-evaporated with toluene to afford36as a pink solid (5.28g,98%).1H NMR(400MHz,DMSO-d6):δ12.7(s,1H), 7.1(s,1H),4.8(s,2H),4.3(q,2H),1.3(t,3H)(method A).

4-(2,6-Di?uoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid (37).A mixture of2,6-di?uorobenzoic acid(6.32g,40.0mmol), 36(5.96g,38.4mmol),EDC(8.83g,46.1mmol),and HOBt (6.23g,46.1mmol)in DMF(100mL)was stirred at ambient temperature for6h.The mixture was reduced in vacuo,water added,and the solid formed collected by?ltration and air-dried to give4-(2,6-di?uoro-benzoylamino)-1H-pyrazole-3-carboxylic acid ethyl ester as the major component of a mixture(15.3g). LC/MS:R t)3.11min,[M+H]+295.Crude4-(2,6-di?uoro-benzoylamino)-1H-pyrazole-3-carboxylic acid ethyl ester(10.2 g)in2M aqueous NaOH/MeOH(1:1,250mL)was stirred at ambient temperature for14h.Volatile materials were removed in vacuo,water(300mL)added and the mixture adjusted to pH 5using1M aqueous HCl.The resultant precipitate was collected by?ltration and dried through azeotrope with toluene to afford 37as a pink solid(5.70g,83%from36).1H NMR(400MHz, DMSO-d6):δ13.60-13.30(br s,1H),10.06(s,1H),8.25(s, 1H),7.68-7.54(m,1H),7.25(t,J)8.4Hz,2H).LC/MS:R t )2.33min(94%purity),[M+H]+267(method A).

4-(2,6-Di?uoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid (trans-4-Amino-cyclohexyl)-amide(27).A mixture of37(100mg, 0.37mmol),(trans-4-amino-cyclohexyl)-carbamic acid tert-butyl ester(98mg,0.46mmol),EDC(86mg,0.45mmol),and HOBt (60mg,0.45mmol)in DMSO(5mL)was stirred at ambient temperature for16h.The mixture was reduced in vacuo,then the residue taken up in CH2Cl2and washed successively with saturated aqueous sodium bicarbonate,water,and brine.The organic portion was dried(MgSO4),?ltered,and the solvent reduced in vacuo.Trituration of the resulting solid with CH2Cl2 gave27,protected with an N-tert-butoxycarbonyl(t-Boc)group. This was removed by treatment with saturated EtOAc/HCl at room temperature for1h.The solid that precipitated out of the reaction mixture was?ltered off,washed with ether,and then dried to give27(14mg,10%).1H NMR(400MHz,Me-d3-OD):δ8.45(s,1H),8.36(s,1H),7.63-7.57(m,1H),7.16(t, J)8.6Hz,2H), 3.91-3.84(m,1H), 3.18-3.11(m,1H), 2.16-2.05(m,4H),2.03(s,1H),1.61-1.50(m,4H).LC/MS: R t)1.75min,[M+H]+364(method A).

Identi?cation of AT7519Journal of Medicinal Chemistry,2008,Vol.51,No.164997

The following compounds were synthesized in an analogous manner to27:

4-(2,6-Di?uoro-benzoylamino)-1H-pyrazole-3-carboxylic Acid Piperidin-4-ylamide(28).4-Amino-piperidine-1-carboxylic acid tert-butyl ester gave28(62mg,48%).1H NMR(400MHz, DMSO-d6):δ13.49(s,1H),10.38(s,1H),9.08(br d,1H),8.69 (br s,2H),8.32(s,1H),7.69-7.58(m,1H),7.27(t,J)8.6 Hz,2H),4.11-3.96(m,1H),3.28(d,2H),3.01-2.88(m,2H), 2.00-1.75(m,4H).LC/MS:R t)1.57min,[M+H]+350 (method A).

4-(2,6-Di?uoro-benzoylamino)-1H-pyrazole-3-(S)-carboxylic Acid Piperidin-3-(S)-ylamide Hydrochloride(29).Using HOAt instead of HOBt,3-(S)-amino-piperidine-1-carboxylic acid tert-butyl ester gave29(55mg,43%).1H NMR(400MHz,Me-d3-OD):δ8.37(s,1H),8.35(s,1H),7.65-7.56(m,1H),7.17(t,J) 8.6Hz,2H),4.32-4.22(m,1H),3.49(dd,J)12.2,4.1,1H), 3.36(s,1H),3.08-2.94(m,2H),2.15-2.02(m,2H),1.92-1.73 (m,2H).LC/MS:R t)1.76min,[M+H]+350(method A). 4-[(4-Amino-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-car-boxylic Acid tert-Butyl Ester(38).

Step 1.A solution of34(7.3g,45.8mmol),4-amino-piperidine-1-carboxylic acid tert-butyl ester(10.2mg,51mmol), EDC(10.7g,55.8mmol),and HOAt(7.5g,55.8mmol)in DMF (100mL),was stirred at room temperature for16h.The solvent was then removed by evaporation under reduced pressure and the residue triturated with water(250mL).The resultant cream solid was collected by?ltration,washed with water,and then dried under vacuum to give4-[(4-nitro-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-carboxylic acid tert-butyl ester(13.05g, 84%).1H NMR(400MHz,DMSO-d6):δ12.53(s,1H),7.67(d, J)8.4Hz,1H),7.10(s,1H),4.60-4.52(m,1H),3.99-3.83 (b m,4H),2.88-2.75(m,2H),1.74-1.68(m,2H),1.52-1.37 (m,11H).LC/MS:R t)2.50min,[M+H]+340(method A). Step 2.4-[(4-Nitro-1H-pyrazole-3-carbonyl)-amino]-piperi-dine-1-carboxylic acid tert-butyl ester(13.05g,38.4mmol)was dissolved in EtOH(300mL)and DMF(75mL),treated with 10%palladium on carbon(500mg)and then hydrogenated at room temperature and pressure for16h.The catalyst was removed by?ltration through celite,the?ltrate evaporated and re-evaporated with toluene.The crude material was puri?ed by ?ash column chromatography eluting with EtOAc then2% MeOH/EtOAc then5%MeOH/EtOAc.Product containing fractions were combined and evaporated to give38(8.78g,74%) as a brown foam.1H NMR(400MHz,DMSO-d6):δ12.53(s, 1H),7.67(d,J)8.4Hz,1H),7.10(s,1H),4.60-4.50(m, 1H),3.98-3.81(m,4H),2.87-2.81(m,2H),1.76-1.66(m, 2H),1.54-1.35(m,11H).LC/MS:R t)1.91min,[M+H]+ 310(method A).

Compound33was Made Using the Following General Method. To a stirred solution of38(0.23mmol),EDC(52mg;0.27 mmol),and HOBt(37mg;0.27mmol)in DMF(5mL)was added the corresponding carboxylic acid(0.25mmol),and the mixture was then left at room temperature for16h.The reaction mixture was evaporated and the residue puri?ed by?ash column chromatography or preparative LC/MS.The compound was treated with saturated EtOAc/HCl and stirred at room temperature for1h.The precipitated solid was?ltered off,washed with ether, and then dried to give the required product.

4-(2,6-Dichloro-benzoylamino)-1H-pyrazole-3-carboxylic Acid Piperidin-4-ylamide Hydrochloride(33).2,6-Dichlorobenzoic acid gave33(64mg,73%).1H NMR(400MHz,Me-d3-O D):δ8.37 (s,1H),7.57-7.43(m,3H),4.22-4.08(m,1H),3.53-3.40(m, 2H),3.22-3.07(m,2H),2.25-2.12(m,2H),1.99-1.80(m, 2H).LC/MS:R t)1.87min,[M+H]+383(method A). Crystallography.Crystals of CDK2were produced using published protocols.47,48Soaking of ligands into CDK2crystals, data collection,ligand?tting,structure re?nement,and structure rebuilding were performed using methods described by Hartshorn et al.1

Biological Assays and DMPK studies.Detailed experimental details can be found in the Supporting Information.

Acknowledgment.We thank Emma Vickerstaffe and Charlotte Grif?ths-Jones for writing the experimental details, Miles Congreve,Martyn Frederickson,Emma Vickerstaffe, Michelle Jones,and Chris Murray for proof reading,and David Rees,Nicola Wallis,and Neil Thompson for useful discussions.We are also grateful to Molecular Imaging Research Inc(Ann Arbor,MI),particularly W.R.Leopold, III,and Erin Trachet,for conducting the xenograft studies using SCID mice.

Supporting Information Available:In vitro kinase selectivity data;detailed descriptions of HPLC methods;HPLC purity analysis of?nal compounds;NMR spectra and HPLC traces for compounds10,12,14,15,18,22,23,28,33;further synthetic chemistry experimental;experimental details for biological assays and DMPK studies.This material is available free of charge via the Internet at https://www.wendangku.net/doc/0117648311.html,.

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如何写先进个人事迹

如何写先进个人事迹篇一：如何写先进事迹材料如何写先进事迹材料一般有两种情况：一是先进个人，如先进工作者、优秀党员、劳动模范等；一是先进集体或先进单位，如先进党支部、先进车间或科室，抗洪抢险先进集体等。无论是先进个人还是先进集体，他们的先进事迹，内容各不相同，因此要整理材料，不可能固定一个模式。一般来说，可大体从以下方面进行整理。 (1)要拟定恰当的标题。先进事迹材料的标题，有两部分内容必不可少，一是要写明先进个人姓名和先进集体的名称，使人一眼便看出是哪个人或哪个集体、哪个单位的先进事迹。二是要概括标明先进事迹的主要内容或材料的用途。例如《王鬃同志端正党风的先进事迹》、《关于评选张鬃同志为全国新长征突击手的材料》、《关于评选鬃处党支部为省直机关先进党支部的材料》等。 (2)正文。正文的开头，要写明先进个人的简要情况，包括：姓名、性别、年龄、工作单位、职务、是否党团员等。此外，还要写明有关单位准备授予他(她)什么荣誉称号，或给予哪种形式的奖励。对先进集体、先进单位，要根据其先进事迹的主要内容，寥寥数语即应写明，不须用更多的文字。然后，要写先进人物或先进集体的主要事迹。这部分内容是全篇材料

的主体，要下功夫写好，关键是要写得既具体，又不繁琐；既概括，又不抽象；既生动形象，又很实在。总之，就是要写得很有说服力，让人一看便可得出够得上先进的结论。比如，写一位端正党风先进人物的事迹材料，就应当着重写这位同志在发扬党的优良传统和作风方面都有哪些突出的先进事迹，在同不正之风作斗争中有哪些突出的表现。又如，写一位搞改革的先进人物的事迹材料，就应当着力写这位同志是从哪些方面进行改革的，已经取得了哪些突出的成果，特别是改革前后的．经济效益或社会效益都有了哪些明显的变化。在写这些先进事迹时，无论是先进个人还是先进集体的，都应选取那些具有代表性的具体事实来说明。必要时还可运用一些数字，以增强先进事迹材料的说服力。为了使先进事迹的内容眉目清晰、更加条理化，在文字表述上还可分成若干自然段来写，特别是对那些涉及较多方面的先进事迹材料，采取这种写法尤为必要。如果将各方面内容材料都混在一起，是不易写明的。在分段写时，最好在每段之前根据内容标出小标题，或以明确的观点加以概括，使标题或观点与内容浑然一体。最后，是先进事迹材料的署名。一般说，整理先进个人和先进集体的材料，都是以本级组织或上级组织的名义；是代表组织意见的。因此，材料整理完后，应经有关领导同志审定，以相应一级组织正式署名上报。这类材料不宜以个人名义署名。写作典型经验材料-般包括以下几部分： (1)标题。有多种写法，通常是把典型经验高度集中地概括出来，一

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城市形态及空间结构分析 ——以郑州市为例城市规划0902 祝相科200917020215 城市形态是城市建设和规划的重要依据，城市规划者的城市形态理念直接决定了城市规划的效果，以致影响城市的总体布局、城市发展的综合效果、交通组织和城镇群的合理分布，甚至关系到城市生产、生活质量、城市改造、城市合理发展方向等一系列重大问题。城市空间是各种人类活动与功能组织在城市地域上的空间投影，是城市建设与发展的载体，城市空间结构的研究是城市发展战略规划的核心内容，城市的区域分析、定位研究以及发展目标的实现等研究最终均要落实到空间上。因此，城市空间结构的合理性对城市的可持续发展也尤为重要。有学者认为城市结构与城市形态互为表里，城市结构表现为城市发展中的内在的动力支撑要素，城市形态则表现为城市发展的外部显性的状态和形式。城市空间结构只是城市结构的一部分，表达的是要素在空间组合上的关系，这种关系即为城市相互作用。城市结构实际上既决定了城市形态，也最终决定了城市空间结构。也就是说，城市空间结构就是能够通过城市相互作用体现为城市形态的那部分城市结构。其实，目前对城市形态并没有形成统一的概念。纵观学者们对城市形态的理解和表述，从横的方面来看，城市形态具有物质和非物质两种表现形式，物质方面主要指城市各有形要素的空间布置方式，包括街道网的结构形式，各种功能的地域分异、城市土地利用模式和建筑环境以及中心城市和相邻城镇群组之间的空间位置关系和结构变化特征等；非物质的主要包括城市生活方式、文化观念和价值观念等所形成的城市社会精神面貌和城市文化特色。从纵的方面来看，城市形态并不是单一的，而是拼贴式的，是各个历史时期的文化积淀的汇合；城市形态不是一成不变的，它会随历史的变化而产生渐进式、碎片式的变化，通过这种渐变，既可以保持城市文化的延续，又能不断地更新。不过每一个不同时期，都会有一种反映时代特色的占主体的城市形态。郑州市城市形态演变过程：郑州市的城市形态经历了“块状发展一点轴延伸一组团分散-一体两翼”的过程，城市的内部填充主要发生于老城区与京广铁路线之间，井向南北方向蔓延。而近20年则是组团分散-一体两翼的转变。并且随着城市规模的扩大，两翼“生长臂”的不断延长，中心城区的中心功能尤其需要不断强化才能满足两翼生长的要求。

结构动力学振型分析

MATALAB 作业某三层钢筋混凝土结构，结构的各层特性参数为：第一层到第三层质量m 分别为2400kg ，1200kg ，1200kg ，第一层到第三层刚度k 分别为3.3*10^4N/m,1.1*10^4N/m,0.66^4N/m.。地震采用acc_ElCentro_0.34g ，采样周期为0.02。 M3=1200kg K3=0.66*10^4N/m. M2=1200kg K2=1.1*10^4N/m M1=2400kg K1=3.3*10^4N/m 用振型分解法求解结构地震反应的MATLAB 层序如下，编制该程序的程序框图以下所示 %振型分解法求解结构地震反应；主程序 clear 开始输入地震参数和结构参数计算结构振型与自振型频率计算振型参与系数计算单自由度体系的地震反应求解结构的地震反应输出结果结束

clc %地震波数据 xs=2*0.287; dzhbo=load('acc_ElCentro_0.34g_0.02s.txt'); ag=dzhbo*0.01*xs; dt=0.02; ndzh=400; cn=3; %cn为结构的层数，即质点数 m0=[2.4 1.2 1.2]*1e+3; %结构各层质量 k0=[3.3 1.1 0.66]*1e+5; %结构各层刚度 l=diag(ones(cn)); m=diag(m0); %计算质量矩阵 [ik]=matrixju(k0,cn); %计算刚度矩阵 [x,d]=eig(ik,m); %结构动力特性求解 d=diag(sqrt(d)); %求解结构圆频率 for i=1:cn; [d1(i),j]=min(d); xgd(:,i)=x(:,j); d(j)=max(d)+1; end %以此循环对所求频率和振型进行排序w=d1; %所求自振频率 x=xgd; %所求结构主振型 a1=2*w(1)*w(2)*(0.05*w(2)-0.07*w(1))/(w(2)^2-w(1)^2); a2=2*(0.07*w(2)-0.05*w(1))/(w(2)^2-w(1)^2); for j=1:cn x(:,j)=x(:,j)/x(cn,j); znb0(j)=(a1+a2*w(j)^2)/2/w(j); zhcan(j)=(x(:,j))'*m*l/((x(:,j))'*m*x(:,j)); %求解振型参数 [dlt(j,:),dltacceler(j,:)]=zxzj(znb0(j),w(j),ag); end %求解结构各层的地震反应 for i=1:cn; disp1=0; accel1=0; for j=1:cn disp0=zhcan(j)*dlt(j,:)*x(i,j); accel0=zhcan(j)*dltacceler(j,:)*x(i,j); disp1=disp1+disp0; accel1=accel1+accel0; end disp(i,:)=disp1; accel(i,:)=accel1; end