Discovery of protease inhibitors using targeted libraries

386

Discovery of protease inhibitors using targeted libraries Mark Whittaker There continues to be considerable effort towards the construction of compound libraries targeted for the inhibition of protease enzymes. New tag-encoding methods for library deconvolution have been applied to this problem and there has been particular interest in novel solid-phase linkers for the introduction of key pharmacophore groups required for protease inhibition. Recent reports have tended to focus on nonpeptidic libraries, and, notably, structure-based design methods are now being applied to direct library design.

Addresses British Biotech Pharmaceuticals Ltd, Watlington Road, Cowley, Oxford, OX4 5LY, UK; e-mail: [email protected] Current Opinion in Chemical Biology 1998, 2:386-396 http://biomednet.com/elecref/1367593100200386 © Current Biology Ltd ISSN 1367-5931

Abbreviations ACE angiotensin-converting enzyme HPLC high performance liquid chromatography MMP matrixMP MMPI MMP inhibitor MP metalloproteinase SAR structure-activity relationship ZBG zinc binding group

Introduction T h e inhibition of protease enzymes has been a target of interest from the outset of the development of combinatorial methods. Initial work was more concerned with technology developments, illustrating their potential by the discovery of inhibitors for readily available, but often not therapeutically relevant, proteases. T h e application of combinatorial chemistry has rapidly become established into tile mainstream of medicinal chemistry and there have been a number of recent reports on its application to the discovery of therapeutically relevant proteascs. For the most part, the approach taken has been to prepare targeted libraries following a 'rational drug design' strategy based on knowledge of the proteolytic mechanism and/or structural information. In many cases, a pharmacophoric group or 'warhead' can be selected that is known to interact with key functionality within the protease active site (for example, statine group for aspartx/l protease inhibitors, aldehyde group for serine and cysteine protease inhibitors and thiol or hydroxamic acid groups for metalloproteinase [MP] inhibitors). Such libraries built around the selected 'warhead' have then addressed the effect of the attached substituents on potency and selectivitx,: For certain therapeutic applications, it is important to obtain selectivity for the inhibition of one particular enzyme within a class (for example, the selective inhibition of thrombin over other serine proteases is

desirable for a therapeutic to be used chronically in stroke patients); however, if the enzymc-inhibitor binding is dominatcd by a potcnt 'warhead', obtaining sclcctivity can bc a difficult task. For many therapeutic applications, it is advantageous to have a compound that not only is a selective inhibitor but is orally bioavailable with a suitable half-life. In the binding of a peptide substrate to a protease enzyme hydrogen bond interactions between the amide groups of the substrate backbone and the enzyme play a significant role. T h e traditional approach to inhibitor design is to attach a 'warhead' to a fragment of a preferred peptide substrate. This results in peptidic-based inhibitors that are highly amenable to preparation by combinatorial methods; however, a disadvantage of such an approach is that it is very difficult to obtain oral activity because the N H group of amides is a known factor in limiting oral absorption. Furthermore, compounds based on natural amino acids may be prone to rapid proteolytic metabolism in virJo and hence have an unacceptably short half-life. Thus, there is increasing interest in tile discovery of nonpeptidic protease inhibitors. This has been achieved in a number of recent examplcs by structure-based design approaches [1 "°] but it is also clear that targeted library methods are now being applied to this problem, often in conjunction with structure-based design [2"]. This review focuses on the advances that have been made in the construction of compound libraries targeted to proteasc inhibitor discovery published in 1997. T h c reader is directed to an excellent review that addresses earlier work in this area [3"]. Combinatorial library methods arc also being applicd to the determination of protcasc substrate selectivity [4,5,P1]. Although these results can be of use in inhibitor design, this work is considered to be outside the scope of this review. Aspartyl protease inhibitor libraries T h e aspartyl protcases renin and HIV protcase have been targets of intensc interest to the pharmaceutical industry [1",6]. A common strategy for the inhibition of this class of proteases is to replace the scissile bond of the substrate with an isostere that mimics tile geometry of the tctrahedral intermediate. Combinatorial methods have been developed following this approach for the preparation of aspartyl protease inhibitor libraries incorporating diamino diol and diamino alcohol cores [7,8]. Recentl> Ellman and co-workers [2",9] reported tile synthesis of two libraries, each of 1,000 compounds, that were prcpared by solid-phase parallel synthesis. T h e libraries were of nonpcptides based on a hydroxycthylamine core (1; Figure 1) and were targeted for the inhibition of cathcpsin D, an aspartyl protease that induces localiscd increases in vascular permcabilitx; fluid accumulation and inflammation. Reaction optimisation enabled thc

Discovery of protease inhibitors using targeted libraries Whittaker

compounds to be obtained in sufficient purity following cleavage such that purification was not required prior to assay. In the first library, substituents were selected to maximise diversity about the hydroxyethylamine core using computational methods; however, more active hits were obtained from a 'directed library' in which the scH)stituents were chosen following a structure-based design approach using the crystal structure of cathepsin I) complexed with the natural peptide inhibitor pepstatin. This second library gave a 'hit rate' of 6-7% at 1 bt.M, as opposed to 2-3% for the diverse library. A second generation library of 39 compounds was then prepared to optimise the most active hits from the 'directed library'. This provided several potent inhibitors of cathepsin D (for example, 2; K i =9n*l) [2"]. ~l\vo recent reports describe the syntheses of hydroxy derivatives that are suitable for the generation of aspartyl protease inhibitor libraries. A solid-phase synthesis of o~-hydroxyketonc derivatives (3, Figure 2) has been achieved involving the immobilisation of an (x-hydroxy ester via the Ellman dihydropyran linker [10] and the reaction of Grignard reagents with the in situ formation of a \Vcinreb amide [11]. A small library of 20 ]3-acetamido carbonyl compounds (4) has been prepared by the solution phase cobah(II) chloride catalysed multicomponent condensation of a ketone, aldehyde and acetonitrile in the presence of acetvl chloride (Figure 2) [121. Serine and cysteine protease inhibitor libraries Many of the known inhibitors of serine and cysteine proteases feature the same types of 'warhead' (for example ct-ketoamide, ]3-lactam, aldehyde), which are able to undergo a covalent interaction with the nucleophilic active site alcohol or thiol group [13-15]. Depending

38?

on the reactivity of the 'warhead' this approach has led to irreversible inhibitors or reversible but tight-binding inhibitors. T h e problem with this approach is that it is difficult to achieve selectivity and so there is considerable interest in the discovery of inhibitors that bind in a noncovalent fashion to the active site. 1600 nonpeptidic ot-keto amide derivatives (5, Figure 3) wer~ prepared by a convergent solution phase parallel synthesis procedure conducted in 96-well plates [16°]. Inhibitors with low micromolar K i values were identified from screening this library against the serine proteases thrombin, factor Xa, trypsin and plasmin. A library of 126 ]3-1actam dipeptides (6, Figure 4) targeted for tile inhibition of human leukocyte clastase, a serine protease implicated in degenerative diseases, has been prepared by parallel solution phase synthesis involving the Ugi multicomponent condensation reaction [17]. A full account has appeared of a solid-phase synthesis ofcarboxy-terminal pcptide aldehydes (7, Figure 5), which involves lithium aluminium hydride mediated cleavage from a \Veinreb amide linker, but this useful process has not been applied to library synthesis [18].

M e t a l l o p r o t e i n a s e i n h i b i t o r libraries The inhibition of MP enzymes has provided effective therapeutics for the treatment of hypertension (inhibition of angiotensin converting enzyme [ACE]) and holds the promise of providing new ways of treating cancer and arthritis (inhibition of matrix methylproteinases [MMPs]) [19,20]. Proteolysis by this class of enzyme involves the activation of the substrate amide carbonyl to attack by water by the active site metal ion, which is usually a zinc(II) ion. T h e principal approach taken to discovering MP inhibitors has been to use a zinc-binding group

Figure 1

o--% o

o,. Cl.~~cI

o

-....~

~ O-!'~

o

o °

Current Opinion in Chemical Biology

From the preparation of two cathepsin D inhibitor libraries (of generic structure 1), each of 1000 compounds, and a smaller follow-up library, potent inhibitors (such as compound 2) were discovered by Ellman and co-workers [2°"].

388

Combinatorial chemistry

Figure 2

(a)

OH RI~N

O ~

(1) R2MgX, THF, RT HCI.HN(OMe)Me

II O

~.

j.O

(2) TFA/DCM/EtOH

(2 (b) ~ N

-{-

O..~H

+

R2 ~./

R3

R2 . ~ I ~ R

CoC,2,AoC,.80oC ~,

R1

O

O

R3 4

1 O

CurrentOpinioninChemicalBiology Reaction schemes for the synthesis of potential aspartyl protease inhibitors. (a) Solid-phase synthesis of o~-hydroxyketonederivatives 3 [11]. (b) The solution phase multicomponent reaction route to ~3-acetamido carbonyl derivatives 4 [12]. DCM, dichloromethane; P, polymer support (polystyrene resin); RT, room temperature; TFA, trifluoroacetic acid; THF, tetrahydrofuran; X, halide.

Figure 3

I IO

(1) HN(R)(CH2)n(R)NH, MeOH, 25oC, 5d

Ar

~_

O~ O

(2)

o

~o

o

RI

~

RI

O ~

O

I/~---Ar'

~N~nN~I~,

Ar o

A~

MeCN or DMFIMeCN, 80oC,24h

CurrentOpinioninChemicalBiology Solution phase parallel synthesis of a 1600-member serine protease inhibitor library of o~-ketoamide derivatives 5 [16"]. Ar, aryl; DMF, dimethylformamide.

(ZBG), such as a thiol, ,¥-carboxyalkyl, carboxylic acid or hydroxamic acid as the 'warhead'. T h e identification of a potent ACE inhibitor (8, Figure 6a) (Ki-160 pM) from a targeted library of 480 proline derivatives that feature a thiol ZBG demonstrated the power of iterative deconvolution to identify active compounds from a library of modest diversity [21]. A recent report from the same group at Affymax describes the use of secondary amine encoding tags [22"] for the direct identification of inhibitor 8 from the same library [23"]. Library preparation involved a key 1,3-dipolar cycloaddition step on solid-phase with tags introduced during the split/pool process. It was concluded that the encoding strategy was

a more efficient means of extracting information from the ACE inhibitor library than iterative deconvolution because it provided structure-activity relationship (SAR) data on a large number of active structures. T h e known ACE inhibitor enaloprilat (9, Figure 6b; R= methyl) was identified as the most active compound in a mixture of 19 compounds using an affinity selection screening technique [24"]. This small targeted library was generated on the solid phase and involved a reductive alkylation step to generate the N-carboxyalkyl ZBG functionality. T h e screening process involved the incubation of the library with ACE followed by size exclusion chromatography to separate unbound corn-

Discovery of protease inhibitors using targeted libraries Whittaker

389

Figure 4

R1

R2

RI~ "1"

O

OH

NH 2

"!"

C~N--R4

R3

/ R2

>,~ MeOH,RT, 18-48h

~.----R4 R3 6

Current Opinion in Chemical Biology

The use of Ugi multicomponent condensation for preparation of a library of ~-Iactam derivatives 6 as potential human leukocyte elastase inhibitors [1 ?]. At, aryl; RT, room temperature.

Figure 5

O Ac-Leu-VaI-Lys(2CI-Z) ~ N . . / . . . . . . . ~

I

N../~ H

OMe

LiAIH4, THF, 0oC

Ac-Leu-VaI-Lys(2CI-Z)-H 7

Current Opinion in Chemical Biology

Solid-phase synthesis of peptidic aldehydes 7 [18]. Ac, acetyl; P, polymer support (polystyrene resin); THF, tetrahydrofuran.

pounds from protein which were then analysed by electrospray ionisation mass spectrometry. This process was then used to identify active inhibitors from the corresponding dipeptide library of 722 compounds in which the carboxy-terminal proline of enaloprilat was varied (for example, Ph(CH2)zCH(COzH)-X1-Xz-OH). T h e N-carboxyalkyl ZBG has also been incorporated into combinatorial libraries targeted against the MMPs [25",26",27]. T h e DuPont-Mcrck group prepared a library of over 100 members using parallel solid-phase synthesis (Figure 7) [25"]. This library was targeted to identify variations of the carboxy-terminal amide substituent of the N-carboxyalkyl library (10, Figure 7). Weak inhibition of MMP-3 (stromelysin-1) was observed (72% inhibition at 200 btM) for the benzhydryl derivative (10; R =CHPh2). In subsequent noncombinatorial studies, the introduction of this modification into succinyl hydroxamic acid derivatives gave more potent compounds [25°]. A 20,000 member library of N-carboxylalkyl tripeptides (11, Figure 8) has been prepared by solid-phase synthesis following a combination of split/pool and indexed techniques [26"°,27]. Again, a reductive amination was used to create the N-carboxyalkyl ZBG functionality (Figure 8). The library was screened against MMP-1 (fibroblast collagenase),

MMP-2 (gclatinase A) and MMP-3 as 100 mixtures each of 200 compounds [27]. Due to the indexed library approach for R 4 and R 3 introduction, SAR information was obtained directly for the P l - P l ' modifications and this was consistent with literature data. Deconvolution resulted in the identification of inhibitors active against each of the enzymes tested (for example, 12 [L-808,457] inhibits MMP-Z and MMP-3 with IC50 values of 14.2btM and 0.2 btM respectively) [27]. Cysteine-containing dipeptides were identified by Glaxo as possessing MMP inhibitor (MMPI) activity and the parallel solid-phase synthesis of compounds RCO-L-CysX-NH 2 led to the identification of inhibitors selective for MMP-1 over MMP-9 (gelatinase B) (for example, CF3CO-L-Cys-L-Phe-NH 2 inhibits MMP-1 and MMP-9 with IC50 values of 40 nM and >1,000 nM respectively) and for MMP-9 over MMP-1 (for example, PhCHzCHzCOL-Cys-L-Phe-NH 2 inhibits MMP-1 and MMP-9 with ICs0 values of 3498nM and 38nM respectively) [28*]. A recent report from the Affymax group [P2] describes the corresponding cysteine-based diketopiperazines (13, Figure 9) that were prepared by solid-phase synthesis on TentaGel TM resin as libraries targeted for MMP inhibition.

390

Combinatorial

chemistry

Figure6 (a) I

%OMe

8

(b)

CO2Et

(1) ~

~

~'°

CO2H R

HOAc-DMF NaBH3CN H-X-Pro-DHPP-PEG-PS (2) TFA-iPrSiH-H20

0

CO2H

9 Current Opinion in Chemical Biology

Angiotensin-converting enzyme inhibitors. (a) Inhibitor 8 was identified from a 480-member library of proline derivatives by iterative deconvolution [21] and by tag-encoding methods [23"]. (b) Solid phase synthesis of N-carboxyalkylderivatives 9 (R, methyl) [24"]. DHPP, 4-(1',1 '-dimethyl-l'-hydroxypropyl)phenoxyacetyllinker; Et, ethyl; Me, methyl; PEG, polyethylene glycol; PS, polystyrene;TFA, tetrahydrofuran.

Figure7

O

O

(1) HCI.NH2CH(CH2CH2Ph)CO2Fm NaCNBH3, DIEA, HOAc-DMF (2) 20% piperidine-DMF (3) HSpfp, DIC, DMF-DCM (4) H2NR, DIEA, HOBt, DMF (5) 4N HCl-dioxane

Me HO'~NII \R O

Current Opinion in Chemical Biology

O 10

Solid phase synthesis of N-carboxyalkyl MMP inhibitors 10 (R, CHPh2) [25"]. DCM, dichloromethane; DIC, 1,3-diisopropylcarbodiimide; DIEA, N,N'-diisopropylethylamine; DMF, dimethylformamide; Fm, 9-fluorenylmethyl; HObt;1-hydroxybenzotriazole;HSpfp, pentafiuorothiophenol; Me, methyl; P, polymer support.

Affymax have explored libraries of MMPIs focused around ZBGs other than thiol including phosphonates [29,30] and more recently carboxylates [31"]. In the latter study, a library of 324 dipeptide succinates (14, Figure 9b) was pre-

pared on TentaGel TM beads following a split/pool protocol using secondary amine encoding tags [22°]. Following photolytic cleavage, the library was screened against matrilysin (MMP-7) in a novel high-density nanowcll

Discovery of protease inhibitors using targeted libraries Whittaker

391

Figure 8

(1) R4COCO2H BH a, pyridine (2) TFA then neutralize (3) 10% Et3N-MeOH, 60°C 0

R2

0

Ra

R4

0

O

0

RI

R2

0

11

H 0

"m oMo

i o 0

~

0

12 Current Opinion in Chemical Biology

A 20,O00-member MMP inhibitor library [26",27]. (a) Solid phase route to N-carboxyalkyl derivatives 11. (b) Inhibitor 12 was identified by deconvolution; Et, ethyl; TFA, trifluoroacetic acid; Me, methyl; P, polymer support.

array format. This study provided direct SAR data for the P2' and P3' amino acids and the resynthesis of two inhibitors led to the identification of one diastereoisomer of (R,S)-3-isobt, tyl-l.-Val-l.-homo-Phe-NH 2 (14; R 1 =iPr, R e =CHzCH2Ph) as a potent MMP-7 inhibitor (IC50 value for inhibition of MMP-7 was 165 nM) [31"]. Rclated succinate inhibitors (15, Figure 10a) have been prepared by parallel-solution phase synthesis using a Ugi multicomponent reaction [P3]. While the products were active as MMP inhibitors (for example, 16 ]Figure 10b] inhil)its MMP-2 with an IC50 value of 60nM); they wcrc converted to the more potent hydroxamic acids (for example, 17 ]Figure 10b] inhibits MMP-1, MMP-2 and MMP-3 with ICs0 values of 600nM, 0.9nM~and 20nM respectively) [32]. T h e transformation to the hydroxamic acids is not a very efficient process and requires product purification by preparative high performance liquid chromatography (HPLC) which, however, does separate

the diastereoisomers. In the special case where a hydroxy group is present alpha to the hydroxamic acid, the desired hydroxamates (18, Figure 1l) were obtained directly in a one-pot five-component condensation [P3,32]. The introduction of the hydroxamate ZBG into succinyl-based MMPIs has been achieved, albeit as a mixture of regioisomers (19 and 20, Figure 12) by the reaction of succinic anhydrides with an O-hydroxylaminetrityl resin (Figure 12) [33]. T h e use of hydroxylamine-presenting resins for the synthesis of MMPIs had been reported earlier [P4,34] and subsequently has been the subject of intense interest [35-39,P5]. In particular, such resins have been utilised in the preparation of a 500-member library of tripeptide hydroxamates Z-X3-Xe-X1-NHOH (where X is any amino acid and Z is benzyloxycarbonyl) [P4] and sulfonamide derivatives [34,39]. A solid-phase Mitsunobu reaction [40] has been employed in the synthesis of the Novartis development sulfonamide hydroxamate (21; CGS27023A; Figure 13) [39]. A diprotected hydroxylamine

392

Combinatorial chemistry

Figure 9

0

(a)

O

(1) R2CHO, TMOF, AcOH, THF, NaCNBH3 (2) Boc-Cys(Trt)-OH, HATU, DIEA, DMF

TG__S__O~'~

NH2

R,

3) 95% TFA-TES, 0.5h 4) 1% AcOH-toluene 12h

H N ' ~

R'

~ N v R HS

~

0 13

(b)

0

.~

0

-"

0

R2

R1

0

14

Current Opinion in Chemical Biology

MMP inhibitor libraries prepared at Affymax (California). (a) Solid phase synthesis of diketopiperzines 13 [P2]. (b) Generic structure of a 324-member dipeptide succinate matrilysin inhibitor library 14 [31 "]. Ac, acetyl; Boc, tert-butoxycarbonyl; DIEA, N,N'-diisopropylethylamine; DMF, dimethylformamide; HATU, 1-hydroxy-?-azabenzotriazole;TES, triethylsilane; THF, tetrahydrofuran; TG, TentaGel resin; TMOF, trimethyl orthoformate; Trt, trityl.

Figure 10

(a)

O ..~

R

O

~

OH

O

(1)R1CHO'NH3'CNR2'MeOH (2) TFA/DCM 1:1

~

R ~

O '

~

= HO

./R2

0

0

RI

15

CI

(b)

0

CI

0

O

O

HO.N le

"~

_

N

04... 17 Current Opinion in Chemical Biology

Succinate MMP inhibitors. (a) Solution phase Ugi multicomponent reaction for the preparation of inhibitor library 15 [P3]. (b) Examplesof MMP inhibitcrs with carboxylic acid (16) and hydroxamic acid (17) ZBG [P3,32]. DCM, dichloromethane; Me, methyl; R, CHPh2; TFA, trifluoroacetic acid.

resin was required to avoid undesired side reactions. A library of novel sulfone MMPIs (22, Figure 14) has been prepared by a process involving parallel synthesis on the solid phase followed by cleavage into solution and resin capture using a O-hydroxylamine presenting

resin [P5]. An alternative method for the solid-phase synthesis of hydroxamic acids involves the derivitisation of an acidic side chain (for example, RCO-Asp(NHOH)-X 1Xz-NH 2) [41]. Finally, from the screening of a large library of tetrapeptides, the compound H-His-eAhx-13Ala-His-OH

Discovery of protease inhibitors using targeted libraries Whittaker

393

Figure 11

o (1)R1CHO,NH3,CNR2,MeOH o

,o,

R

O

NTy

(2) 48h then NH2OH

N OH

O

R~

18 Current Opinion in Chemical Biology

One pot-five component synthesis of o~-hydroxyhydroxamicacid MMP inhibitors 18 [P3]. Me, methyl.

Figure 12

R

(1) oo ~ - ' o , --O--NH 2

o

60°C,THF

R

O

•

(2) H2NR2,HOBt,DCC,DIVIF 0

R

0

(3) HCO2H-THF Current Opinion in Chemical Biology

Solid phase synthesis of regioisomeric succinyl hydroxamicacid MMP inhibitors 19 and 20 [33]. DCC, 1,3-dicyclohexylcarbodiimide; DMF, dimethylformamide; P, polymer support; THF, tetrahydrofuran.

Figure 13

(1) DIC,Fmoc-D-VaI-OH,DMF (2) 4-MeOCsH4SO2CI,NMM,DCM (3) Me2N(O)CN=NC(O)NMe2,Bu3P, D

I (~

N'--.H

3-pyridylCH2OH,THF (4) 2.5% TFA, 1% H20 in DCM (5) 50% TFA, 1% H20 in DCM

/ j ~

" ~ " N ~

OMe

"°'N 21

Current Opinion in Chemical Biology

Solid-phase synthesis of the Novartis MMP inhibitor 21 [39]. DIC, 1,3-diisopropylcarbodiimide;Fmoc, 9-fluorenylmethoxycarbonyl;DMF, dimethylformamide; Me, methyl; NMM, N-methylmorpholine; DCM, dichloromethane; Me, methyl; THF, tetrahydrofuran; TFA, trifluoroacetic acid; P, polymer support (TentaGel S resin).

(where Ahx is 6-amino caproic acid) was identified as a weak inhibitor of M M P - 2 and M M P - 9 (IC50 values for inhibition were 400 laM and 300 btM, respectively) [42].

Conclusions It is clear that targeted library m e t h o d s are an effective means for discovering protease inhibitors especially when

394

Combinatorial chemistry

Figure 14

O

(1) (EtO)2POCH2CO2H,DMF, pyridine,2,6-diCIC6H3COCI ""OH (2) a. KHMDS,THF,toluene b. R1CHO

Wang

O

R1

(1) ~

' ' ' ' ' O / N H2

(1) R2SH,nBuLi,THF HO"

(2) mCPBA (3) TFA-DCM (1:1) O NO

"~N"

v

"S

EDCI,DMF (2) TFA-DCM (1:1)

R1 v

-S /

22 Current Opinionin ChemicalBiology Solid-phase synthesis, incorporating resin capture, of sulfone MMP inhibitors 22 [P5]. DCM, dichloromethane; DMF, dimethylformamide; EDCI, N-ethyI-N-(3-dimethylaminopropyl)carbodiimide; Et, ethyl; KHMDS, potassium hexamethyldisilazide; mCPBA, meta-chloroperoxybenzoic acid; P, polymer support (polystyrene resin); R1, iPr; R2, CH2CH2Ph; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

used in conjunction with strncture-based design methods [2••,25",431. To date, the use of solid-phase s'~'nthesis has predominated over solution based methods for the preparation of targeted libraries. Key strategic decisions in the synthetic planning are the attachment point to the resin and whether to use parallel or split/pool methods. Attachment via the 'warhead' group as used in the synthesis of 1, 3, 10 and 21 (Figures 1,2,7 and 13) enables greater diversity of substitution. Conversely, attachment elsewhere should allow the generation of libraries to discover novel 'warheads' but this approach has not been explored to date. An advantage of parallel synthesis is that completc SAR data is obtained; however, it is now clear that similar data can be obtained from the first round of screening of split/pool libraries either by tag encoding [23",31 "°] or indexing methods [26°°]. Although in some early reports on protcase inhibitor libraries the compounds werc screened while still attached to the solid phase [30,44], in all the reported in vitro library testing covered in the review the compounds were screened in solution. It has recently been demonstrated, however, that the serine protease trypsin can cyclise certain p o l y m e r - s u p p o r t e d peptidic amino esters and it is suggested that this will provide a new paradigm for e n z y m e inhibitor discovery from combinatorial libraries [45"'].

Acknowledgements The author is grateful to Andrew Ayscnugh, Paul Beckett and Chris Floyd

for their comments on this review"and to Jenny I)nllard for assistance in preparation of the manuscript.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • *.

of special interest of outstanding interest

1. •.

Babine RE, Bender SL: Molecular recognition of protein-ligand complexes: applications to drug design. Chem Rev 1997, 97:1359-1472. A thorough review on structure-based design that encompasses aspartyl, serine, cysteine and matrix metalloproteinase enzyme inhibitors. 2. •*

Kick EK, Roe DC, Skillman AG, Liu G, Ewing TJA, Sun Y, Kuntz ID, EIIman JA: Structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin D. Chem Bio11997, 97:297-307 This account of the identification of potent inhibitors of the aspartyl protease cathepsin D illustrates the merit of utilising a structure-based design approach for the selection of substituents within a targeted combinatorial library. 3. Dolle RE: Discovery of enzyme inhibitors through combinatorial •. chemistry. Mol Divers 1996, 2:223-236. This key review summarises the literature (up to and including most of 1996) on the use of combinatorial libraries in the identification of both protease inhibitors and compounds active against nonproteolytic enzymes. 4.

Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP et aL: A combinatorial approach defines specificities of members of the caspase family and granzyme B. J Biol Chem 1997, 272:17907-17911.

5.

Rano TA, Timkey T, Peterson EP, Rotonda J, Nicholson DW, Becker JW, Chapman KT, Thornberry NA: A combinatorial approach for determining protease specificities: application to interleukin-l[3 converting enzyme (ICE). Chern Biol 1997, 4:149-155. Thaisrivongs S: HIV protease inhibitors. Annu Rep Med them 1994, 29:133-144.

Discovery of protease inhibitors using targeted libraries Whittaker

Kick EK, Ellman JA: Expedient method for the solid-phase synthesis of aspartic acid protease inhibitors directed toward the generation of libraries. J Med Chem 1995, 38:1427-1430.

395

hibition. The advantage of this technique over iterative deconvolution is that structure-activity relationship data can be obtained from single bead assays.

8.

Wang GT, Li S, Wideburg N, Krafft GA, Kempf DJ: Synthetic chemical diversity: solid phase synthesis of libraries of C 2 symmetric inhibitors of HIV protease containing diamino diol and diamino alcohol cores. J Med Chem 1995, 38:2995-3002.

Blackburn C, Pingali A, Kehoe T, Herman LW, Wang H, Kates SA: Libraries of angiotensin converting enzyme inhibitors: solidphase synthesis and affinity selection. Bioorg Med Chem Lett 1997, 7:823-826. The use of affinity selection in conjunction with mass spectroscopy as described in this preliminary account provides a method for screening large mixtures of compounds prepared by combinatorial methods.

9.

EIIman J, Stoddard B, Wells J: Combinatorial thinking in chemistry and biology. Proc Nat/Acad Sci USA 1997, 94:2?792782.

25. •

10

Thompson LA, EIIman JA: Straightforward and general method for coupling alcohols to solid supports. Tetrahedron Lett 1994, 35:9333-9336.

11.

Wallace OB: Solid phase synthesis of ketones from esters. Tetrahedron Lett 1997, 38:4939-4942.

12.

Mukhopadhyay M, Bhatia B, Iqbal J: Cobalt catalyzed multiple component condensation route to #-acetamido carbonyl compound libraries. Tetrahedron Lett 1997, 38:1083-1086.

13.

Hlasta DJ, Pagani ED: Human leukocyte elastase inhibitors. Annu Rep Med Chem 1994, 29:195-204.

14.

Ripka WC, Vlasuk GP: Antithrombotics/serine proteases. Annu Rep Med Chem 1997, 32:71-89.

15.

Otto H-H, Schirmeister T: Cysteine proteases and their inhibitors. Chem Rev 1997, 97:133-1 71.

16. •

Batdino CM, Casebier DS, Caserta JC, Slobodkin G, Tu C, Coffen DL: Convergent parallel synthesis. Synlett 1997:488490. A publication from the Arqule group illustrating that parallel solution phase methods can provide a practical alternative to solid-phase methods for targeted library synthesis. 1 '7.

Pitlik J, Townsend CA: Solution phase synthesis of a combinatorial monocyclic 13-1actam library: potential protease inhibitors. Bioorg Med Chem Lett 1997, 7:3129-3134.

18.

24. •

Rockwell A, Melden M, Copeland RA, Hardman K, Decicco CP, DeGrado WF: Complementarity of combinatorial chemistry and structure-based ligand design: application to the discovery of novel inhibitors of matrix metalloproteinases. J Am Chem Soc 1996, 118:10337-10338. Potent MMP inhibitors were identified by utilising a targeted library together with structure-based design. The approach used here of subsequent structural analysis of active inhibitors identified from library screening is somewhat different to the discovery of cathepsin D inhibitors by EIIman and co-workers [2"'], who used structural information to direct the library design. 26. •.

Esser CK, Kevin NJ, Yates NA, Chapman KT: Solid phase synthesis of a N-carboxylalkyl tripeptide combinatorial library. Bioorg Med Chem Lett 1997, 7:2639-2644. This paper illustrates that a combined split/pool and indexed library strategy can lead to useful structure-activity relationship data from the initial screen of a targeted library. 2?.

Kevin NJ, Esser CK, Chapman KT, Hagmann WK, Yates NA, Kostura MJ, Pacholok SG, Si Q: The synthesis of a 4dimensional N-carboxymethyl peptide combinatorial library for new lead generation against metalloproteinases utilizing both mix and split and indexed library strategies. Abstract MEDI 113 of the 213th ACS National Meeting: 1997 13-17 April; San Francisco. Washington DC: American Chemical Society; 1997.

28. •

Foley MA, Hassman AS, Drewry DH, Greet DG, Wagner CD, Feldman PL, Berman J, Bickett DM, McGeehan GM, Lambert MH, Green M: Rapid synthesis of novel dipeptide inhibitors of human collagenase and gelatinase using solid phase chemistry. Bioorg Med Chem Lett 1996, 6:1905-1910. This report describes the parallel synthesis of potent inhibitors of MMP-1 and MMP-9 that feature a thiol ZBG. 29.

Campbell DA, Bermak JC: Solid-phase synthesis of peptidylphosphonates. J Am Chem Soc 1994, 116:6039-6040.

Fehrentz JA, Paris M, Heitz A, Velek J, Winternitz E Martinez J: Solid phase synthesis of C-terminal peptide aldehydes. J Qrg Chem 1997, 62:6792-6796.

30.

Campbell DA, Bermak JC, Burkoth TS, Patel DV: A transition state analogue inhibitor combinatorial library. J Am Chem Soc 1995, 117:5381-5382.

19.

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20.

Davidson AH, Drummond AH, Galloway WA, Whittaker M: The inhibition of matrix metalloproteinase enzymes. Chem Ind 1997:258-261.

21.

Murphy MM, Schullek JR, Gordon EM, Gallop MA: Combinatorial organic synthesis of highly functionalized pyrrolidines: identification of a potent angiotensin converting enzyme inhibitor from a mercaptoacyl proline library. J Am Chem Soc 1995, 117:7029-7030.

Ni Z-J, Maclean D, Holmes CP, Murphy MM, Ruhland B, Jacobs JW, Gordon EM, Gallop MA: Versatile approach to encoding combinatorial organic syntheses using chemically robust secondary amine tags. J Med Chem 1996, 39:16011608. The first report of the Affymax single bead encoding methodology that involves the use of an N-[(dialkylcarbamoyl)methyl] glycine coding oligomer. Decoding involves acidolytic secondary amine formation, dansylation and analysis by reverse phase high performance liquid chromatography.

Schullek JR, Butler JH, Ni Z-J, Chen D, Yuan Z: A high-density screening format for encoded combinatorial libraries: assay miniaturisation and its application to enzymatic reactions. Anal Biochem 1997, 246:20-29. This paper describes the preparation of a library of MMP-7 inhibitors that were screened in a novel miniaturised format and identified by tag encoding. 32.

Whittaker M, Floyd CN, Leblanc C J, Lewis CN, Miller A, Saroglou L, Patel S: Parallel synthesis of matrix metalloproteinase inhibitors. Abstract 76 of the 7th International Kyoto Conference on New Aspects of Organic Chemistry: 1997 10-14 November: Kyoto. Kyoto: IKCOC; 1997.

33.

Bauer U, Ho W-B, Koskinen AMP: A novel linkage for the solidphase synthesis of hydroxamic acids. Tetrahedron Lett 1997, 38:7233-?236.

34.

Floyd CD, Lewis CN, Patel SR, Whittaker M: A method for the synthesis of hydroxamic acids on solid phase. Tetrahedron Lett 1996, 37:8045-8048.

35.

Richter LS, Desai MC: A TFA-cleavable linkage for solid-phase synthesis of hydroxamic acids. Tetrahedron Lett 1997, 38:321322.

36.

Gordeev MF, Hui HC, Gordon EM, Patel DV: A general and efficient solid phase synthesis of quinazoline-2.4-diones. Tetrahedron Lett 1997, 38:1729-1732.

37.

Mellor SL, McGuire C, Chan WC: N-Fmoc-aminooxy-2chlorotrityl polystyrene resin: a facile solid-phase methodology

22. •

23. •.

Maclean D, Schullek JR, Murphy MM, Ni Z-J, Gordon EM, Gallop MA: Encoded combinatorial chemistry: synthesis and screening of a library of highly functionalized pyrrolidines. Proc Nat/Acad Sci USA 1997, 94:2805-2810. This is an important paper because it demonstrates the utility of the Affymax secondary amine tag encoding methodology in the identification of an active inhibitor from a library targeted for angiotensin-converting enzyme in-

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for the synthesis of hydroxamic acids. Tetrahedron Lett 1997, 38:3311-3314. 38.

Mellor SL, Chan WC: 4-[2,4-dimethoxyphenyl(N-fluoren-9ylmethoxycabonyI-N-alkylaminooxy)-methyl]phenoxymethyl polystyrene: a multiple solid phase approach to Nalkylhydroxamic acids. J Chem Soc Chem Commun 1997:20052006.

39.

Ngu K, Patel DV: A n e w and efficient solid phase synthesis of hydroxamic acids. J Org them 199?, 62:7088-7089.

40.

Dankwardt SM, Smith DB, Porco JA, Nguyen CH: Solid phase synthesis of N-alkyl sulfonamides. Synlett 199?:854-856.

41.

Chen JJ, Spato[a AF: Solid phase synthesis of peptide hydroxamic acids. Tetrahedron Lett 1997, 38:1511-1514.

42.

43.

44.

Ferry G, Boutin JA, Atassi G, Fauchere J-L, Tucker GC: Selection of a histidine-containing inhibitor of gelatinases through deconvolution of combinatorial tetrapeptide libraries. Mol Divers 1996, 2:135-146. Salemme FR, Spurlino J, Bone R: Serendipity meets precision: the integration of structure-based drug design and combinatorial chemistry for efficient drug discovery. Structure 1997, 5:319-324. Bastos M, Maeji NJ, Abeles RH: Inhibitors of human heart chymase based on a peptide library. Procl Nat/Acad Sci USA 1995, 92:6738-6742.

45. •*

Burger MT, Bartlett PA: Enzymatic, polymer-supported formation of an analog of the trypsin inhibitor A 9 0 7 2 0 A : a screening strategy for macrocyclic peptidase inhibitors. J Am Chem Soc 1997, 119: 12697-12698. In this study, trypsin is used to catalyse the reverse (amide synthesis) reaction for a polymer-supported peptidic amino ester. Structures that cyclised were distinguished from those that did not cyclise by the inclusion of a base labile ester bond within the sequence. Treatment with base resulted in the loss of a dye-labelled fragment from noncyclised structures but beads for which the cyclisation occured remained coloured. The structures of the cyclised sequences could be used as the basis for inhibitor design.

Patents P1.

Quibell M, Johnson T, Hart T: Substrates and inhibitors of proteolytic enzymes. 30 October 1997, WO9740065.

P2.

Campbell DA, Look GC, Szardenings AK, Patel DV: Metalloproteinase inhibitors. 24 December 1997, WO9748685.

P3.

Floyd CD, Whittaker M: Synthesis of carboxylic and hydroxamic acid derivatives. 6 September 1996, WO9626918.

P4.

Floyd CD, Lewis CN: Synthesis of hydroxamic acid derivatives. 29 August 1996, WO9626223.

P5.

Groneberg RG, Neuenschwander KW, Djuric SW, McGeehan GM, Burns C J, London SM, Morrissette MM, Salvino JM, Scotese AC, UIIrich JW: Substituted (awl, heteroaryl, arylmethyl or heteroarylmethyl) hydroxamic acid compounds. 10 July 1997, WO9724117.