Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets

Preparation of Kinase-BiasedCompounds in the Search for Lead

Inhibitors of KinaseTargets

Justine Y.Q. Lai, Steven Langston, Ruth Adams, Rebekah E. Beevers,Richard Boyce, Svenja Burckhardt, James Cobb, Yvonne Ferguson,

Eva Figueroa, Neil Grimster, Andrew H. Henry, Nawaz Khan, Kerry Jenkins,Mark W. Jones, Robert Judkins, Jeremy Major, Abid Masood, James Nally,Helen Payne, Lloyd Payne, Gilles Raphy, Tony Raynham, John Reader,

Valerie Reader, Alison Reid, Parminder Ruprah, Michael Shaw,Hannah Sore, Matthew Stirling, Adam Talbot, Jess Taylor,

Stephen Thompson, Hiroki Wada, David Walker

Millennium Pharmaceuticals R&D Limited, Granta Park, Great Abington, Cambridge CB1 6ET,

United Kingdom

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20026

!

Abstract: This work describes the preparation of approximately 13,000 compounds for rapid

identification of hits in high-throughput screening (HTS). These compounds were designed as

potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds,

e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the

hydrogen bond acceptor/donor properties known to be important for kinase binding. Several

of these are based upon literature kinase templates, or adaptations of them to provide novelty.

The routes to their preparation are outlined. A variety of automation techniques were used to

prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to

remove excess reagents andwhen necessary, preparative high performance liquid chromatography

(HPLC) was used for purification. These compounds were screened against an ‘in-house’ kinase

panel. The success rate in HTS was significantly higher than the corporate compound collection.

� 2004 Wiley Periodicals, Inc. Med Res Rev, 25, No. 3, 310–330, 2005

Key words: lead generation; kinase inhibitors; parallel synthesis; high-throughput screening

JustineY.Q.Lai is aprimaryauthor.

Correspondence to: Steven Langston, Millennium Pharmaceuticals, 40-2 Landsdowne Street, Cambridge, MA 02139, USA.

E-mail: [email protected]

Medicinal Research Reviews, Vol. 25, No. 3, 310^330, 2005

� 2004 Wiley Periodicals, Inc.

1 . I N T R O D U C T I O N

Protein kinases have emerged as attractive targets in the search for new therapeutic agents. Their

inhibition has proven an attractive strategy in several therapeutic areas, particularly oncology and

inflammation.1 The use of structural information obtained by available X-ray structures of protein

kinases, in many cases complexed with inhibitors, as well as computer-assisted molecular modeling

based on kinase domain homology has allowed the design of selective protein kinase inhibitors.

This has resulted in advances in small molecule kinase inhibitors into clinical trials, with some

notable successes such as Glivec2 and Iressa.3

We recently embarked on a campaign to provide kinase inhibitor-biased compounds to

supplement the corporate compound collection and be used to rapidly identify hits in primary

screening, especially against future kinase targets. High-throughput screening (HTS) has tradi-

tionally yielded low hit rates. It was envisaged that a focused library would significantly improve

the hit rate. The particular kinase targets are not necessarily known at this stage, and a review of the

literature1,2a suggests that targeting the adenosine triphosphate (ATP) binding site might give broad-

spectrum tyrosine, or serine/threonine kinase inhibitors. Selectivity can then be achieved depending

upon the construct of the particular kinase active site. The library was, therefore, designed to contain

compounds exhibiting as many known or potential binding modes for ATP as possible.

2 . L I B R A R Y D E S I G N

The library was designed according to a core template that generally includes the hydrogen bond

acceptor/donor properties known to be important for kinase binding. Several of these libraries are

based upon literature kinase inhibitor templates, and some on adaptations of these to provide novelty.

Some of the scaffolds are summarized in Figure 1.

For each core template, a virtual library was generated using a selection of 200–300 building

blocks at each point of diversity. These building blocks were either commercially available or

prepared in-house. Prior to synthesis, the physicochemical properties of a virtual library of these

compounds were calculated using software from the Accelrys CombichemConsortium.4 Monomers

were chosen to give a library of 2,500–4,000 compounds, most of which exhibit drug-like properties.

A typical profile for the AlogP and number of rotatable bonds for the whole of the virtual library and

the proposed synthetic library is shown (Fig. 2). These show that the mean AlogP decreases from 3.9

Figure 1. Summaryof library templatesusedbasedonknownkinase scaffolds.

PREPARATION OF KINASE-BIASED COMPOUNDS * 311

to 2.6, with 94% of the synthetic library being less than 5. The mean number of rotatable bonds

decreases from 10 to 7, with 87% having 8 or fewer rotatable bonds. From this the monomers were

further shortlisted by the chemist on practical grounds such as costs, availability, and solubility as

well as chemical variety (e.g., acidic and basic groups, H-bond donor and acceptor groups, alkyl and

aryl groups) at each point of diversity. This final selection also ensured that all the molecules passed

Lipinski’s rules5 as well as having a polar surface area (PSA)6 <140A2.

The synthesis was planned to yield a minimum of 5 mg of final compound with purity>70% by

evaporative light scattering (ELS) detection.A brief introduction to the background7 of each template

and their parallel synthesis is described in the following section. The success rates quoted for the

syntheses, unless otherwise stated, are for compounds that met the required yield and purity criteria.

3 . A Z A N A P H T H A L E N E S

This is a broad template, based on several known inhibitors of different classes of kinases (Fig. 3).

In the quinazoline class of compounds, PD 153035was first reported in 1994 as an epidermal growth

factor receptor (EGFR) kinase inhibitor.8 This has led to the preparation of a large number of

analogs.1a,7a,7d The Food and Drug Administration (FDA) has granted accelerated approval9 to

AstraZeneca’s Iressa3 as a single agent treatment for patients with advanced non-small-cell lung

cancer, and Iressa has been approved in Japan and Australia. A new major development of the

quinazoline class has been the replacement of the N-3 atom by a nitrile groupwithout a sharp drop off

in EGFR potency.1a Manipulation of the aniline substitution pattern has allowed the identification of

a potent and selective Src inhibitor10 and MEK (MAP kinase kinase) inhibitor.11 The quinoxaline

class of compounds, as exemplified byRPR 101511A andRPR 127963, are platelet-derived growthfactor receptor (PDGFR) kinase inhibitors, which display excellent selectivity against other receptor

kinases.1a The azanaphthalene sub-templates are shown in Figure 4.

Using Template A as representative, preparation of these classes of compounds is summarized in

Scheme 1. Parallel synthesis was carried out in solution phase in a H&P reaction block,12 which was

Figure 2. Example of some of the physico-chemical properties of the virtual library and all the planned compounds in a typical

library template.

312 * LAI ET AL.

linked to the TECANNeptune13 instrument to allow automated dispensing of reagents. The chloride

was displaced by alkoxides (generated from alcohols and sodium hydride) and amines. The

displacement using primary and secondary alkyl amines was carried out in refluxing EtOH. For aryl

or heteroaryl amines, elevated temperatures were required. This was carried out either in DMSO

at 80�C in a Micronics plate or N-methyl-2-pyrrolidinone (NMP) at 150�C in an H&P block.

Purification was carried out by aqueous work-up and preparative high performance liquid

chromatography (HPLC). This synthesis had a 61% success rate, yielding >1,400 compounds.

4 . P Y R I M I D I N E S

Compounds derived from pyrimidine template have been screened for inhibition of a variety of

protein kinases, such as Abelson leukaemia virus (ABL),2 cyclin-dependent kinase (CDK),14 and

EGFR7a (Fig. 5). Glivec2 is the first kinase inhibitor to be approved by the FDA (in 2001). The

pyrimidine sub-templates are shown in Figure 6.

In Templates A and B, preparation of this class of compounds is exemplified in Scheme 2.

Parallel synthesis was carried out using either the advanced Chemotech (ACT) or Irori Kans.15 Prior

to synthesis, the resin bound amines were prepared by reductive amination of primary amine

monomers onto formyl resin (4-formyl-3-methoxyphenoxy resin).16 Thesewere then treated with an

Figure 4. Azanaphthalene library sub-templates.

Figure 3. Knownquinazoline, quinoxaline, andquinolinekinase inhibitors.

Scheme 1. Reagents and conditions: (i) R1OHor R1NH2,iPr2EtN,EtOH, 80

�C, 48 h.


excess of the symmetrical 4,6-dichloropyrimidine in NMP at 80�C in the presence of iPr2NEt to give

the 4-amino-derivatives. The second chloride was displaced with piperazine. The free secondary

amino group was then further reacted to yield sulfonamides (using sulfonyl chlorides), ureas (using

isocyanates), and amides (using acyl chlorides). Alternatively, they were reacted with aldehydes

under reductive conditions to yield tertiary amines. The products were cleaved from resin at ambient

temperature using 50% trifluoroacetic acid (TFA) in CH2Cl2. Synthesis of template Cwas carried out

using palladium coupling.17 Overall, this synthesis had a 63% success rate, yielding >2,500

compounds. In this template, due to issues using Irori Kans (see General Remarks section), 70% of

the compounds did not yield the required quantities. Nevertheless sufficient amounts were isolated to

enableHTSof these final products.Hence the success rate quoted for this template is for yields of 2–3

mg of final compound with the required purity. (See Scheme 2.)

Figure 5. Knownpyrimidinekinase inhibitors.

Figure 6. Pyrimidine library sub-templates.

Scheme 2. Reagents and conditions: (i) NMP,80�C,18h (ii) piperazine,NMP,100�C,18h (iii) (a) RCHO,DMF (1%AcOH), rt,18h

(b) NaBH(OAc)3, DMF (1%AcOH), rt,18 h (iv) RSO2Cl,iPr2NEt,DMAP,CH2Cl2, rt, 7 h (v) RNCO,CH2Cl2, rt, 7 h (vi) RCOCl, iPr2NEt,

CH2Cl2, rt,7 h.

314 * LAI ET AL.

5 . P U R I N E S

Purine analogs have been screened for inhibition of a variety of protein kinases.7a,b,d Examples of

purines as kinase inhibitors include Olomoucine7a and Purvalanol,7a which are CDK inhibitors

(Fig. 7).7a

The purine sub-templates are shown in Figure 8.Using Template B as representative, preparation

of this class of compounds is summarized in Scheme 3.18 Parallel synthesis was carried out using

ACT 49619 automation apparatus, and any necessary purification was carried out using HPLC.

In a similar fashion to the purine template, the resin bound amines were prepared by reductive

amination of primary amine monomers onto a formyl resin prior to synthesis.16 These resin-bound

amines were then treated with an excess of dichloropurine in NMP at 80�C in the presence of iPr2NEt

to displace the chloro- group at the 6-position. The second chloridewas displaced by a primary amine

using higher temperatures as this is not as activated as the 6-position chloride. Alkylation at the 9-

position was effected by treatment with an alkyl halide in the presence of the tertiary amine base, 1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU), Scheme 3. Cleavage from resin using TFA in CH2Cl2furnished the final product. As there is precedence for the parallel synthesis of these compounds,7a,18

a number of more speculative monomers were examined. In some of these cases, alkylation took

place at the 7-position in addition to the 9-position. Furthermore, displacement of the second chloro

group at the 2-position using a-substituted primary amines could not be easily achieved despite

the harsh conditions. As a result, this synthesis had only a modest success rate (41%), nevertheless

yielding >800 compounds.

6 . I M I D A Z O L E S

This template is broadly based on P38MAP kinase inhibitors,20 as exemplified by SB 203580, which

was the first member of this compound class to be reported as a kinase inhibitor (Fig. 9).

As such, the synthesis of arrays of imidazoles was sought Key steps to their preparation via

keto-amide 3 as the key intermediate is shown in Scheme 1. Keto-amides 3 are prepared using the

carbazate linker 1. After cleavage, cyclization of the keto-amide 3 was effected by treatment with

NH4OAc under elevated temperatures21 to furnish the imidazole 4 (Scheme 4).

Figure 7. Knownpurinekinase inhibitors.

Figure 8. Purine library sub-templates.


The keto-moiety of the tertiary amide is essential for cyclization of the imidazole. Hence, a

general solid-phase synthesis approach to display diverse functionality about a ketone would enable

the preparation of libraries of keto-amides and subsequently imidazoles. The library template of

1,2,4-trisubstituted is shown in Figure 10.

The linkage of a ketone to a solid support using the carbazate linker has been described by

Ellman.22 Carbazate linker 1 was prepared from commercially available polystyrene-poly(ethylene

glycol) resin (ArgoGel-OH, Argonaut Technologies) by coupling with hydrazine using 1,1 0-carbonyldiimidazole (CDI). It was reported that 60 equivalents of anhydrous hydrazinewere required

to provide near quantitative loading levels (0.3–0.4 mmol/g).22 The use of hydrazine monohydrate

was examined and the reaction followed by FTIR analysis.23 It was observed that similar loadings

could be obtained with only 10 equivalents of hydrazine monohydrate (Scheme 5), demonstrating

that anhydrous conditions were not essential.

Retrosynthetic analysis of keto-amide 3 is shown in Scheme 6. From the analysis, it was also

evident that cleavage of the intermediates to monitor the synthetic route would not be conclusive,

as this would require determining mass recovery of the a-chloroketone 8 from the cleavage of

hydrazone 7, or isolation of an unstable aminoketone from cleavage of 6. Hence the synthesis would

be taken through to the resin-bound hydrazone amide 2 before cleavage to examine the overall yield

and purity (Scheme 6).

A dehydration reaction with a-chloro-ketone gave the a-chloro-hydrazone. The validation

study (Scheme 4) was carried out using 2-chloro-4 0-fluoroacetophenone, benzylamine, and 4-

bromophenylacetic acid. Condensation was investigated at 45�C and room temperature. After

aminolysis, acylation, and cleavage, the condensation at 45�Cgave quantitativemass return in>85%

purity,24 whereas condensation at room temperature gave slightly reduced yield (85%) and purity

(80%). Different solvents for the condensation reaction were also examined. It was observed

that whilst tetrahydrofuran (THF) and dimethylformamide (DMF) gave similar results, dioxane gave

poorer yields (30–40% less by comparison). The limited commercial availability of a-chloroketones

Scheme 3. Reagents and conditions: (i) iPr2NEt,NMP, 80�C,8 h (ii) R3NH2,NMP,150�C,12 h (iii) R2X,DBU,NMP, 20

�C,12 h (iv)TFA,CH2Cl2, 20

�C, 2� 30min.

Figure 9. Known imidazolekinase inhibitor.

316 * LAI ET AL.

led to further exploration of this step. One option was the preparation of a-chloroketones from the

corresponding a-methylketones.24 Alternatively, it was noted that this condensation was also

applicable to a-bromoketones, even though they yielded 30–50% less product when directly

compared with the corresponding a-chloroketones. Nevertheless the ability to use a-bromoketones

in the synthesis further increased the choice for monomer selection in the library production

(Scheme 7).

While the previous stepwas effected in the fumehood, parallel synthesis of subsequent steps was

carried out using the ACT 49625 or Vantage25 automation apparatus. Displacement of the chloride

was effected by use of excess primary amine. The resultant secondary amine was acylated with a

carboxylic acid.Acid coupling conditionswere examined using eitherHOAt orHOBt in combination

with PyBOP, or HATU. All gave similar results. Hence HOBt and PyBOP were selected due to

availability and economic reasons.

Cleaveage of the ketoamide from the resin was successfully carried out using a mixture of

1:4:4:15 TFA/H2O/MeCHO/TFE, as reported by Ellman. MeCHO is utilized as a hydrazine

scavenger.22 However, the volatility of the carcinogen MeCHO meant that its safe removal on a

library production scale would not be feasible. Direct replacement of MeCHOwith acetone resulted

in successful cleavage by analysis of the keto-amides.26 Typically, the purity of these compounds

were >95% by ELS.

The final step was carried out in solution phase, whereby the ketoamide was cyclized to the

imidazole using ammonium acetate in acetic acid. Overnight treatment of the keto-amide with a

saturated solution of NH4OAc in AcOH and DMF (6:1) at 90�C furnished the desired 1,2,4-

trisubstituted imidazoles, Scheme 8. The excess NH4OAc was removed by two methods, depending

on the substituent R1. When R1 ¼ aryl, an aqueous work-up and re-extraction of the product into

EtOAc was carried out. When R1 ¼ alkyl, this method was not applicable as the higher aqueous

Scheme 4.

Figure 10. Imidazole library template.

Scheme 5. (i) CDI,CH2Cl2, rt, 3 h (ii) H2NNH2.H2O,DMF, rt,1h.


solubility of the products resulted in poor compound recovery after aqueous extraction. To

circumvent this, the excess NH4OAc was removed by freeze-drying (Scheme 8).

In general from resin-bound a-chloro carbazate 7, the imidazoles were obtained over four steps

in >30% yield and >95% purity (Table I).

Overall preparation27 of these compounds is summarized in Scheme 9. Purification of some

compounds was carried out by preparative HPLC. This synthesis had a 65% success rate, yielding

>1,400 compounds (Scheme 9).

7 . O X I N D O L E S

This class of compounds was first published in 1994 as ABL kinase inhibitors.28 This template seems

to be very versatile, exhibiting significant inhibition of various kinases (VEGFR, FGFR1, PDGFR,

EGFR, HER-2, CDK, ABL, p38, lck, and JNK).1a The first clinical candidate in this class came from

Sugen. SU 5416 (semaxanib) is a potent inhibitor of vascular endothelial growth factor receptor

(VEGFR) in cells and underwent phase 3 clinical trials.29 (See Figs. 11 and 12.)

Preparation30 of these compounds is summarized in Scheme 10. Parallel synthesis was carried

out in solution phase inMicronics plates and FlexchemBlocks.31A range of oxindolemonomerswith

various substitutions on the aromatic ring were either bought in or prepared in-house. So as to drive

the reaction to completion, these monomers were reacted with a slight excess (1.5 eq) of aldehyde in

Scheme 6.

Scheme 7. (i) 2-Chloro-4 0-fluoroacetophenone,THF, 45�C, 4 h (ii) benzylamine, DMF, rt, 30 min (iii) 4-bromophenylacetic acid,PyBOP,HOBt,DiPEA,DMF, rt, 2�4 h (iv) 1:4:4:15 TFA:H2O: acetone:TFE, rt, 2� 2 h.

318 * LAI ET AL.

the presence of piperidine base. Removal of any remaining aldehyde was carried out using

aminomethyl polystyrene scavenger resin. After filtration of the resin, products were obtained in

excellent purity. This synthesis had a tremendous success rate (83%), yielding >1,500 compounds

(Scheme 10).

8 . A M I D O - H E T E R O C Y C L E S

There was in-house precedence to suggest that these would be potential kinase inhibitors, where the

peptidic side chain was shown to be essential for potency. Consequently, a library was proposed

focusing on the peptide side chain, with a lesser diversity on the heteroaromatic ring (Fig. 13).

Preparation of these classes of compounds is summarized in Scheme 11. Stratified addressable

chemistry (StAC)32 parallel synthesis using standard peptide coupling was carried out. StAC is a

technique that relies on the use of modular solid support, such as Mimotope SynPhaseTM L-series

lanterns (a grafted plastic support).33 This method was developed in-house, where the use of the

lanterns allowed stacking, thereby generating a three-dimensional ‘‘plate.’’ Typically, approximately

four to five layers of 8� 12 plates are used, potentially yielding 384–480 compounds per run.

Scheme 8. (i) 6:1sat.NH4OAc (AcOH):DMF, 90�C, 24 h.

Table I. Yield and Purity Data for Representative Analogs from the Imidazole Library

aDeterminedbyELS.

bOverall yieldofsolidandsolutionphasereactions.


Nopurificationwas required on these compounds. This synthesis had an excellent success rate (83%),

yielding >1,000 compounds.

9 . I S O Q U I N O L I N E S U L F O N A M I D E S

Isoquinolinesulphonamides, e.g.,H89, have been reported to be potent inhibitors of protein kinase C

(PKC)34 and cAMP-dependent protein kinase.35 The N on the isoquinoline ring is key to binding,

whilst the rest of the heteroatoms also aid inH-bond interactionswithin theATPpocket either directly

or indirectly (via a water molecule).34 (See Figs. 14 and 15.)

Preparation36 of these compounds is summarized in Scheme 11. It was envisaged that arrays of

these compounds would be prepared from the respective sulfonyl chloride and amine. Hence novel

quinoline and isoquinoline sulfonyl chlorides would provide useful building blocks for enhancing

novelty as well as structural diversity. In this series, monomer preparation involved chlorosulfony-

lation of the starting quinoline/isoquinoline. This was effected by chlorosulfonic acid. Isolation of

the sulfonyl chloride was achieved either in a one-step procedure (Method B) or in two-steps via

initial isolation of the sulfonic acid (Method A). The isolation of the sulfonic acid was identified by

the characteristic stretch in the infra-red (umax 2,800–2,000/cm). These were then converted to the

sulfonyl chloride in a subsequent step using thionyl chloride (Method A) (Table II).

Scheme 9. Reagents and conditions (i) (a) CDI,CH2Cl2, rt, 3 h (b) H2NNH2.H2O,DMF, rt,1h (ii) a-chloroketone,THF, 45�C, 4 h(iii) R1NH2, DMF, 20

�C, 30 min (iv) acid, PyBop, HOAt, iPr2NEt, DMF, 20�C, 2�4 h (v) TFA:H2O:acetone:TFE (1:4:4:15), 20�C, 4 h

(vi) NH4OAc, AcOH, 90�C, 24 h.

Figure 11. Knownoxindolekinase inhibitor.

Figure 12. Oxindole library template.

320 * LAI ET AL.

Polymer-supported dimethylaminopyridine (PS-DMAP)37 has been used for trapping sulfonyl

chlorides, which then react with amines to the corresponding sulfonamide.38 Parallel synthesis was

carried out using this ‘‘resin-capture and release’’ method on the FlexchemBlocks.39 In the first step,

the sulfonyl chloride was captured onto PS-DMAP. This activated species was then cleaved using an

amine to generate the final product. Purification, where necessary, was carried out by preparative

HPLC. This synthesis had a 60% success rate, yielding >650 compounds (Scheme 12).

A. Representative Preparation Procedure of Sulfonamide from the CorrespondingSulfonyl Chloride

DMAP on polystyrene resin (100 mg, 3.75� 10�2 mmol, 5eq) was suspended in CH2Cl2 (1 ml) and

shaken for 5 min and then drained. A solution of the sulfonyl chloride (10eq) was added to the resin

and shaken for 45 min. The excess reagents were drained, and each well washed with CH2Cl2(3 times). A solution of the amine (1eq), DiPEA (10eq) in CH2Cl2 was added. The resultant mixture

was allowed to shake at rt for 20 h. The reactionwas then drained andwashedwith CH2Cl2 (2� 1ml).

The filtrate was concentrated under vacuo to yield the desired sulfonamide.

A further 520 compounds (68% success rate) were prepared using the StAC technology,32 using

a route similar to that previously described.39

1 0 . I M I D A Z O P Y R I D I N E S

There is no precedence that this class of compounds would be potential kinase inhibitors. However,

they have a structural resemblance to the purine class of kinase inhibitors. As such, it was proposed

as a potentially novel kinase template (Fig. 16).

Scheme 10. Reagents and conditions: (i) R1CHO, piperidine,DMF/EtOH, 80�C, 5 h.

Figure 13. AmidoHeterocycle library template.

Scheme 11. Reagents and conditions: (i) (a)R1NH2,DMF, (2%AcOH), rt,1h (b)NaBH(OAc)3,DMF(2%AcOH), rt,18h(ii)aminoacid,PyBrOP,

iPr2NEt,DMF, rt, (iii) piperidine (20%),DMF, rt, 2�1h (iv) acid,PyBroP, iPr2NEt,DMF, rt, (v) TFA (20%),CH2Cl2, rt1h.


Preparation40 of this class of compounds is summarized in Scheme 13. In the first step, 2,3-

dichloropyrazine was treated with ammonium hydroxide in a pressure reactor. In the subsequent

steps, parallel synthesis was carried out in solution phase using Techne Dri-Block Heaters.41 The

resultant 2-amino-3-chloropyrazine was reacted with an a-bromoketone to yield the chloro-

imidazopyrazine. Displacement with a range of amines followed by scavenging of excess amine

using PS-isocyanate furnished the final compounds in good purity. This synthesis had an outstanding

success rate (89%), yielding >400 compounds.

1 1 . G E N E R A L R E M A R K S

This campaign to provide kinase-biased inhibitor compounds to supplement the corporate compound

collection for rapid identification of hits in primary screening was successfully concluded with

�13,000 compounds prepared. Several of these libraries mentioned are from the first generation of

the campaign, where they are based upon literature kinase templates. Despite potential patentability

Figure 14. Known isoquinolinesulfonamidekinase inhibitor.

Figure 15. Isoquinolinesulfonamide library template.

Table II. Chlorosulfonylation of Quinoline and Isoquinoline Using ClSO3H

MethodA: (i)ClSO3H,20�C,2h (ii) SOCl2,80

�C,2h.

MethodB:ClSO3H,100�C,18h.

*Step (i) heated to100�C,18h.

322 * LAI ET AL.

issues, it is generally believed that they will provide a useful starting point from HTS for hitherto

unknown kinases. In the second generation of kinase-biased libraries, some adaptations of known

scaffolds have been designed to provide novelty and patentability. The imidazopyridine scaffold is an

example of this production.

In the preparation of these compounds, a variety of techniques were utilized (see Table III),

with different scopes and limitations. In general, an average of 3 months was required for validation

of the chemistry and transfer to library production. Extra time in validation tends to increase the

success rate of the library, although this is usually balanced out by time that could have been used for

going into production.

One of the more powerful tools for parallel synthesis of large numbers of compounds is the

use of Irori Kans. Within a set time frame, they allow the preparation of three to four times more

compounds than the other equipment utilized for solid phase chemistry. However, some limitations

were observed during production of the libraries. Whilst the miniKans can hold more resin,

movement of the tag within the Kan during agitation led to leakage and grinding of the resin, thereby

leading to loss of compound. In addition, this technology is less amenable to heterogenous reactions

where the vigorous agitation required often resulted in extensive leakage of resin. As a result,

furnishing the required quantities of compounds can be challenging. Flexchemblocks are useful tools

for short syntheses, due to the need for manual handling. They are also useful for scavenging excess

reagents. Automation using the ACT is useful for multi-step solid phase chemistry, as it is a one-stop

synthetic technique. Addition of reagents and solvents, washing of resin in intermediate steps, and

final cleavage can all be programmed. Typically, production of a multi-step library involved several

days of continuous automation. One limitation is the rate of sequential addition of reagents

and solvents over 96-well reactor plates. The fact that reactions carried out manually on the ACT

reactor plates gave a comparable rate of library production to using automation is indicative of this

Figure 16. Imidazopyridine library template.

Scheme 13. Reagents and conditions: (i) NH4OH,130�C, 20 h (ii) a-bromoketone, iPr2NEt,

iPrOH, 85�C,18 h (iii) R3R4NH, EtOH,

80�C,18 h.

Scheme 12. Reagents and conditions: (i) (a)PS-DMAP,CH2Cl2,20�C,45min(b)R1HN(CH2)nNR2R3,

iPr2NEt,CH2Cl2,20

�C,20h.


Table

III.

SummaryofTem

plates,theTechniques

Used,andOutput

(Continued

)

324 * LAI ET AL.

Table

III.

(Continued

)


drawback. StAC is regarded as valuable new technology that essentially allows multiple plates to be

prepared per production run, thereby increasing the efficiency in the long run.

Equipment for parallel synthesis in solution phase allows a wide range of temperatures.

However, these do not allow the preparation of asmany compounds at any one time. Themost utilized

equipment across the project was the Tecan Neptune liquid handler. It allows dispensing of reagents

for solution phase chemistry, and can be programmed to heat reactions.Whenever necessary, aqueous

work-up can also be carried out.

In most cases, synthesis of each library can be carried out using more than one technique.

In general, solution phase techniques work well with syntheses that involve one to two steps only.

The use of solid phase is more amenable to routes that contain more steps; a variety of factors can

influence the final selection. The choice whether to carry out the synthesis in solid or solution phase

largely depends on precedence. From here, preference for a particular technique is based on

temperature and scale of reaction, synthetic route, post-reaction work-up as well as the robustness of

the chemistry to yield the required amounts at the end. No single technique is all encompassing.

1 2 . H I G H - T H R O U G H P U T S C R E E N I N G

The 13,000 (approximately) compounds prepared were incorporated as a subset of the corporate

compound collection for HTS against various targets. The data are summarized in Table IV.

The actives from the HTS are defined as compounds whose activities have been reconfirmed in an

assay. Although the kinase-biased library subset is <15% of the corporate collection, comparatively

it had a significantly improved hit-rate. For example, when screened against Kinase 1, the corporate

collection (>200,000) had a 1% success rate of actives. In contrast, a 9%success ratewas obtained for

the kinase-biased subset of compounds. Unsurprisingly, results against the other targets Non-Kinase

1 (an enzyme with an ATP-binding site) and Non-Kinase 2 (a GPCR), did not show any advantage

over the corporate collection. For Kinase 5, even though the percentage was lower compared with

the other kinases, it was encouraging that the IC50 of the compounds were in the nanomolar range

when measured. (See Table IV.)

Table IV. High-Throughput Screening Results of Compounds in Corporate

Collection Versus Kinase-Biased Library Collection

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1 3 . C O N C L U S I O N

Approximately 13,000 kinase-biased compounds were prepared using a variety of equipment/

techniques. This was successfully achieved over 15 months using an average of 14 FTE in synthesis.

These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors, using

known kinase inhibitors or their adaptations as templates. A mixture of solid-phase (ACT, Irori,

Flexchem Block) and solution-phase (Tecan, H&P block, Micronics Plate) techniques were utilized.

The compounds were incorporated as a subset of the corporate compound collection for HTS against

various targets. In kinase targets, a significantly improved hit-rate (>2.5-fold) was obtained over the

corporate collection.

1 4 . A B B R E V I A T I O N S

ABL v-abl, Abelson leukemia virus tyrosine kinase

ACT advanced ChemTech

ATP adenosine triphosphate

CDK cyclin-dependent kinase

DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

DMAP dimethylaminopyridine

EGFR epidermal growth factor receptor

ELS evaporative light scattering

FDA food and drug administration

Fyn fyn, p59fyn

GPCR G-protein coupled receptor

HPLC high performance liquid chromatography

HTS high-throughput screening

Lck lymphoid T-cell protein tyrosine kinase, lck, p60lck

MAP mitogen-activated protein

MEK MAP kinase kinase

NMP N-methyl-2-pyrrolidinone

P38 p38 mitogen activated protein kinase

PDGFR platelet-derived growth factor receptor

PKA protein kinase A

PKC protein kinase C

PS polymer-supported

PSA polar surface area

SRC Rous sarcoma virus oncoprotein, c-src, pp60c-src

StAC stratified addressable schemistry

TFA trifluoroacetic acid

VEGFR vascular endothelial growth factor receptor

A C K N O W L E D G M E N T S

The authors gratefully acknowledge the efforts of the following people: Analytical and Purification:

Allan Muir, Jo Ridgway, Krystyna Holden, Liz Wood, Pahee Siva, Paula Bryans; Automation:

John Humphries, Craig Grant, Dan Cowell, Graeme Langlands, Matthew Jones, Suki Klair, William

Wilson.


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Justine Lai received her BSc (Hons) and Ph.D. in organic chemistry at Imperial College, London, working with

Prof. Donald Craig. She joined Rhone-Poulenc Rorer (now Aventis Pharma) in 1996 as a medicinal chemist,

working in pharmaceutical drug discovery in the asthma and inflammation therapeutic areas. She moved to

Millennium Pharmaceuticals in 2000, where she was initially involved in high-throughput synthesis and

subsequently medicinal chemistry within the Oncology Discovery Chemistry Department.

Steve Langston received his BSc (Hons) and Ph.D. in organic chemistry from Sheffield University under the

guidance of Prof. Mike Blackburn before undertaking a post-doctoral fellowship with Prof. A. Vasella at the

E.T.H., Zurich. He joined Peptide Therapeutics (nowMedavir) in 1996 developing combinatorial approaches to

identifying protease inhibitors. He joined Millennium Pharmaceuticals in 2000 and is a medicinal chemist

within the Oncology Discovery Chemistry Department.

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Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets

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