combinatorial peptide and nonpeptide libraries || synthesis and evaluation of three...

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung

0 VCH Verlagsgesellschaft mbH, 1996

15 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

Barry A. Bunin, Matthew J. Plunkett and Jonathan A. Ellman

15.1 Introduction

One of the initial steps in the development of therapeutic agents is the identification of lead compounds that bind to the receptor or enzyme target of interest. Many analogs of the lead compounds are then synthesized to define the key recognition elements for maximal activity. In general, many compounds must be evaluated in both the lead identification and optimization steps. Recently, the demand for compounds for drug discovery efforts has increased dramatically. This is due in large part to recent technological advances in screening procedures for many therapeutic targets that allow for the rapid and efficient evaluation of thousands to millions of compounds. To address this demand, very powerful chemical and biological methods have been

developed for the generation of large combinatorial libraries of peptides and oligonucleotides that are then screened against a receptor or enzyme to identify the high affinity ligands or potent inhibitors, respectively [I]. While these studies have clearly demonstrated the power of combinatorial synthesis and screening strategies, peptides and oligonucleotides generally have poor oral activities and rapid in vivo clearing times, which limit their utility as bioavailable therapeutic agents. As a result of such limitations, the synthesis and screening of libraries of nonbiological oligomers [2, 31, and most recently nonpolymeric organic compounds 14, 51, have rapidly become the focus of intensive research efforts. Derivatives of 1,Cbenzodiaze- pines have widespread biological activities, and are one of the most important classes of bioavailable therapeutic agents [6]. Benzodiazepine derivatives have been reported that act as anxiolytic, anticonvulsant, and antihypnotic agents [8], selective cholecystokinin (CCK) receptor subtype A or B antagonists 171, K-selective opioid antagonists [8], platelet activating factor antagonists [9], HIV trans-activator Tat antagonists [lo], GPIIbIIIa inhibitors 1111, reverse transcriptase inhibitors 1121, and ras farnesyl transferase inhibitors [ 13).

Because of the broad biological activity and desirable pharmacokinetics of 1,4-benzodiazepine derivatives, we have developed general and expedient solid-phase synthesis methods for this class of molecules. Here, we describe the methods used for the design and simultaneous synthesis of three benzodiazepine libraries that incorporate a wide variety of chemical functionality.

406 I5 Synthesis and Evaluation of Three 1,4-Bentodiazepine Libraries

15.2 Synthesis Criteria for a Benzodiazepine Library

In the construction and evaluation of a library of 1,4-benzodiazepine derivatives, we felt that several criteria should be met. (i) The benzodiazepine derivatives should be synthesized on a solid support because the solid support strategy allows for facilc isolation of polymer-bound reaction products from reagent mixtures. This enable: one to drive reactions to completion by the use of excess reagents. (ii) The variable components, or building blocks, used for the synthesis of a benzodiazepine librarj should be readily synthesized or (ideally) commercially available. This greatly ex pedites the process of library synthesis, since time is not consumed in the repetitive synthesis of different building block derivatives. (iii) After synthesis of the compounds is complete, the compounds should be removed from the support so that the compounds can be assayed in solution because the solid support may complicate or interfere with receptor binding to the support-bound small molecule. (iv) Initially, in the construction of the library, the compounds should be synthesized in a spatially separate fashion to enable rigorous chemical and biological characterization of the library. In contrast to solid-phase peptide and oligonucleotide synthesis, general methods for the solid-phase synthesis of organic compounds have, until recently, seen limited development 114, IS]. When new solid-phase synthesis methods are employed, the chemical integrity and relative yields of library members can readily be determined when compounds are spatially separate. Also, by maintaining the compounds spatially separated, biological evaluation often provides detailed structure-versus-activity data. (v) The construction of a library of organic compounds that relies upon techniques already developed for high throughput screening procedures avoids the development of new instrumentation.

15.3 Chiron Mimotopes (Geysen) Pin Apparatus

In constructing the libraries, we have employed the Chiron Mimotopes pin apparatus, originally developed by Geysen for peptide epitope mapping 116, 171. In this apparatus, 96 polyethylene pins are placed into a supporting block so that each pin fits into a separate well of a 96-well microtiter plate. The pins are prederivatized with aminoalkyl groups, providing sites for substrate attachment, and each well of the microtiter plate serves as a distinct reaction vessel for performing chemical reactions. Currently, pin loading levels that range from 100 nmol to 50 pmol of material per pin are available. Even 100 nmol of material is sufficient for multiple biological assays, as well as for analytical evaluation of the purity and chemical integrity of the individual compounds.

While our libraries synthesized to date have been made using the Geysen pin apparatus, our development of solid-phase synthesis methods has generally been performed on crosslinked aminomethvl Dolvstvrene resin. which allows for vield deter-

15.4 Solid-Phase 1,4-Benzodiazepine Synthesis 407

minations based on mass balance. Because optimization has been performed on gel- form resin, our synthetic methods may readily be adapted to a library synthesized with a split-and-mix approach initially developed by Furka [18] and subsequently ex- panded by many others 1191.

15.4 Solid-Phase 1,4-Benzodiazepine Synthesis

Because of the broad biological activity and favorable pharmacokinetic properties of the 1,4-benzodiazepines, we set out to develop general methods for the synthesis of libraries of this class of molecules [20]. The first step in the synthesis of an organic compound library is to develop general reaction conditions that allow a variety of different functionality to be incorporated in high yield. The 1,6benzodiazepine derivatives were initially constructed from three components: 2-aminobenzophenones, amino acids, and alkylating agents [21]. Employing solution chemistry, substituted 2-N-Fmoc-aminobenzophenones are coupled to the acid cleavable 4-hydro- methylphenoxyacetic acid (HMP) linker. As shown in Scheme 15-1, the linker may be attached through a hydroxyl or carboxyl group located on either aromatic ring of the 2-aminobenzophenone. The linker derivatized aminobenzophenones 1 are then coupled to the solid support by standard amide bond-forming methods.

l a 2a

l b

Scheme 15-1. 2b

Synthesis of benzodiazepine derivatives on solid support (Scheme 15-2) is initiated by removal of the Fmoc protecting group from 2 by treatment with piperidine in DMF. An a-N-Fmoc amino acid is then coupled to the resulting unprotected 2-aminobenzophenone. Standard activation methods for solid-phase peptide synthesis were not successful for this coupling step due to the poor basicity and nucleophilicity of 2-aminobenzo~henones. However, the activated a-N-Fmoc amino acid fluorides

408 15 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

developed by Carpino [22] couple efficiently to provide the amide products 3 even for electron deficient 2-aminobenzophenone derivatives. The Fmoc protecting group is then removed using piperidine in DMF, and the resulting free amine is treated with 5 % acetic acid n NMP or DMF at 60°C to provide the benzodiazepine derivatives 4 that incorporate two of the three components for introducing diversity.

NHFmoc

1. piperidine c

2. 2. 5% AcOH. NMP

NHFmoc

2 3

0 Li‘

TFA

4 5 6

Scheme 15-2.

Alkylation of the anilide of 4 provides the fully derivatized 1,Cbenzodiazepines 5. In order to ensure complete reaction on solid support, excess reagent is generally employed. We therefore employ either lithiated 5-phenylmethyl-2-oxazolidinone or lithiated acetanilide as the base since they are basic enough to completely deprotonate the anilide of 4, but will not deprotonate other functionality that may be pre- sent in the benzodiazepine structure such as amide, carbamate, or ester functionality. Treatment with the volatile acid cleavage cocktail trifluoroacetic acid/dimethyl sulfide/H20 (85 : 10 : 5 ) then affords the benzodiazepine products 6, which after chromatography are obtained in high yield (85- 100%) based on the support-bound starting material 2. Finally, no racemization (c 1 TO) of selected derivatives was detected as determined by chiral HPLC. Structures of representative compounds prepared by this route are shown in Fig. 15-1.

15.4 Solid-Phase 1,4-Benzodiazepine Synthesis 409

O H OH

Figure 15-1. Structures of representative 1.4-benzodiazepine derivatives prepared by the route of Scheme 15-2. The average yield for the derivatives shown is 90'70, based upon the initial 2-aminobenzo~henone loading level of the resin.

410 I5 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

15.5 First Generation 1,4-Benzodiazepine Library

With the development of general solid-phase strategy for benzodiazepine synthesis, we set out to synthesize and evaluate a benzodiazepine library in order to evaluate the spatially separate synthesis of these compounds [23]. Using the Chiron Mimo- topes pin support, a library of 192 compounds was assembled using ail combinations of two 2-aminobenzophenones, twelve amino acids, and eight alkylating agents, with a variety of functionality being displayed (Fig. 15-2).

The chemical integrity and yield of many of the compounds in the library were determined by two analytical methods. For 28 of the structurally diverse benzodiazepine derivatives, FAB mass spectrometry confirmed the structure of the compound corresponding to the major peak (in almost all cases the only peak) observed

2-Aminobenzophenones H2N 0 H2N 0 8^0-& \ OH HO \

CI

Amino Acids

H H

Alkylating Agents

-' /I \ I -1

H'

Figure 15-2. Building blocks used in the synthesis of first generation benzodiazepine library (192 spatially separate compounds).

15.7 Current Solid-Phase 1,4-Bentodiazepine Synthesis 41 1

by HPLC. Yields were also determined for 20 derivatives, where each of the 2-aminobenzophenones, amino acids and alkylating agents was incorporated in at least one of the derivatives. This was accomplished by addition of a stock solution containing fluorenone as an internal standard followed by HPLC analysis. An 86% average yield for the benzodiazepine derivatives was observed as calculated from the experimentally determined extinction coefficients of the selected derivatives.

The spatially separate library of benzodiazepines was screened to identify ligands to the cholecystokinin A receptor using a competitive radioligand binding assay. Detailed structure versus activity information was obtained for this receptor target. The data provided by screening the library was confirmed by synthesizing a number of the derivatives on large scale followed by purification and IC,, determinations. The most potent compound (K5, = 0.08 p ~ ) was synthesized from 2-amino-4-hy- droxybenzophenone, D-tryptophan, and ethyl iodide. In addition, comparison of our structure-activity data with that obtained by Merck researchers for a series of structurally related benzodiazepines showed close correlation.

15.6 Second Generation 1,4=Benzodiazepine Library

With the successful synthesis and evaluation of the initial 192 member benzodiazepine library, the same strategy was used to synthesize a larger library for screening against a variety of targets. This second generation library was synthesized from three 2-aminobenzophenones, 35 amino acids, and 16 alkylating agents, providing 1680 1,4-benzodiazepine derivatives (Fig. 15-3). With this library we have identified inhibitors of pp60c”R: tyrosine kinase [24] and ligands that block an autoimmune DNA-antibody interaction [25] implicated in systemic lupus erythematosus. These results suggest that a modestly sized library based upon an appropriate template can often be sufficient to identify ligands or inhibitors.

15.7 Current Solid-Phase 1,4-Benzodiazepine Synthesis

The original benzodiazepine synthesis sequence was based upon the combination of three different building block sets: a 2-aminobenzophenone, an Fmoc amino acid fluoride, and an alkylating agent. While many alkylating agents are commercially available, and N-Fmoc amino acid fluorides can be prepared in a single step without purification from the corresponding N-Fmoc amino acids, few appropriately functionalized 2-aminobenzophenones are readily accessible. To increase the diversity of 1,4-benzodiazepine-2-ones available through solid-phase synthesis, we utilized the Stille coupling reaction to synthesize a variety of 2-aminoaryl ketones on solid support. The Stille reaction is particularly appealing for this purpose since it proceeds


2-Aminobentophenones H2N 0

Hm OH CI JyQ ’ ’ OH CI OH

Amino Acids (Both Enantiomers Incorporated)

Alkylating Agents H* /I W I W‘ -‘ &er

Figure 15-3. Building blocks used in the synthesis of second generation benzodiazepine library (1680 sDatially separate comDounds).

15.8 Design of a Large 1,4-Bentodiazepine Library 41 1

under mild conditions, is tolerant of a wide range of functionality, and well over 300 structurally diverse and chemically compatible acid chloride building blocks are commercially available.

The support-bound 2-(4-biphenylyl)isopropyloxycarbonyl (Bpoc) protected aminoarylstannane 7 (Scheme 15-3) is prepared in six steps from commercially available material. Coupling to support is again accomplished employing the HMP linker. Stille coupling can be carried out with a range of different acid chlorides and the catalyst Pd2(dba)3 .CHC13. Because excess acid chloride is employed in the Stille coupling step, diisopropylethylamine and K2CO3 are added as acid scavengers to minimize protodestannylation. The Bpoc group is cleaved by brief treatment with 3% TFA in CH2C12, and the resulting support-bound 2-aminoaryl ketone 8 is then incorporated directly into 1,4-benzodiazepine derivatives using the previously described synthesis sequence 1261.

6 7 Scheme 15-3.

9

Using this strategy, numerous acid chlorides were employed to prepare support- bound 2-aminoaryl ketones 8 that were further incorporated into 1 ,Cbenzodiaze- pines 9, including aromatic acid chlorides that are electron rich, electron poor, alkyl substituted, polyaromatic, heterocyclic, and orthosubstituted, and aliphatic acid chlorides that can be sterically hindered. The desired benzodiazepines were isolated after the eight-step synthesis sequence in > 85% purity by 'H NMR analysis of the crude products. Yields of purified benzodiazepine products varied from 52 070 to 82% (Table 15-2) based on the initial aminomethyl loading of the polystyrene resin used. The structures of represantative compounds are shown in Fig. 15-4. The full characterization and mass balance based yields of the products obtained demon- strate that a wide variety of functional groups are compatible with this solid-phase synthesis methodology.

15.8 Design of a Large 1,4-Benzodiazepine Library

With a versatile procedure for the solid-phase synthesis of 2-aminobenzophenones and 2-aminoacetophenones in hand, we designed the synthesis of a large library of structurallv diverse 1.4-benzodiaze~ine derivatives. Since this librarv is to be evalu-

414 I5 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

Meal$

0

OMe

Figure 15-4. Structures of representative 1.4-benzodiazepine derivatives prepared by the Stille coupling route of Scheme 15-3. The average yield for the derivatives shown is 70V0, based upon the initial aminomethyl substitution level of the resin.

ated against a number of different therapeutic targets, we wanted to display a wide range of chemical diversity about the rigid benzodiazepine scaffold, thereby maxi- mizing the possibility of finding lead compounds for each of these targets. A search of the Available Chemicals Directory (ACD) [27] for chemically compatible building

15.9 Synthesis of an 11 200 Member 1,4-Benzodiazepine Library 41

blocks shows that over 300 acid chlorides, 80 Fmoc-protected amino acids, and 800 alkylating agents can be purchased. If all of these components were included, the library would contain well over 19 million compounds! The component that are available and compatible do not realistically limit the ultimate size of a benzodiazepine library. Because our previous experience has shown that a library of less than 2000 benzodiazepine derivatives is sufficient to find ligands for several different medicinal targets, we decided to set the size of the third generation library at approximately IOOOO compounds.

We chose to generate a 1,Cbenzodiazepine library containing 11 200 derivatives, which would be prepared from 20 acid chlorides, 35 amino acids [28], and 16 alkylating agents (Fig. 15-5). These numbers were chosen in part so that compounds in the library would divide neatly and rationally into the wells of a microtiter plate. Natural and unnatural amino acids containing amines, amides, carboxylic acids, alcohols, phenols, thiophenes and indoles were included. Alkylating agents containing a range of aromatic and aliphatic groups, as well as alkylating agents with hydrogen bond donors and acceptors were incorporated. The acid chlorides were selected with assistance from a structural similarity procedure developed by Steven Muskal at MDL Information Systems [29]. A list of over 500 commercially available acid chlorides was pared to approximately 350 based solely on the predicted chemical compatibility of each acid chloride with the benzodiazepine synthesis sequence. The acid chlorides were then grouped; structurally similar derivatives were placed into the same bin and structurally different acid chlorides were in separate bins. From the resulting 45 bins [30], 20 diverse acid chlorides were chosen for inclusion in the library. Generally, only the least substituted acid chloride was chosen from a par- ticular bin. The components for each of the three building blocks were selected based on commercial availabilitiy and maximal structural diversity, to generate an 1,4-benzodiazepine library displaying a wide range of chemical functionality.

15.9 Synthesis of an 11 200 Member 1,4-Benzodiazepine Library

With a diverse set of building blocks in hand, we proceeded to synthesize the third generation benzodiazepine library [31]. This benzodiazepine library includes derivatives with indole, phenol, ether, cyclohexyl, heterocyclic, polyaromatic, halogen, thiophene, furan, cyano, carboxylic acid, amine, amide, and hydroxyl functional groups. The development of reliable solid-phase synthtic methods is the rate deter- mining step in library synthesis. The actual construction of this library of 11 200 compounds took two graduate students about one month each, and could be faster with automation.

The majority of the building blocks, or structurally similar building blocks like ethyl- and hexyl-alkylating agents, were incorporated into fully characterized

41 6 I5 Synthesis and Evaluation of Three 44-Benzodiazepine Libraries

Acid Chlorides 0 CI

I ,'N /

CI &OMe

0

CN

CI

Amino Acids

Alkylating Agents H+ /I V I / G B r - 1 o '

NC-1

Figure 15-5. Set of structurally diverse reagents used for the synthesis of the third generatior 1,4-benzodiazepine library (11 200 compounds).

15.10 Alternate Strategies for Benzodiazepine-Based Diversity 411

1,4-benzodiazepines synthesized on large scale. Assuming an untested building block will be compatible with a combinatiorial synthesis is risky. For example, the 1,4-benzodiazepines derived from cyclopropyl carbonyl chloride, with structural homology to the 1,4-benzodiazepines derived from cyclohexyl carbonyl chloride (previously synthesized and characterized on beads) [16], contained side products in addition to the expected structure.

Approximately 100 nmol of racemic 1,4-benzodiazepine was obtained in each well after removal of the cleavage cocktail. Ten aliquots of this library were made from a single synthesis, to be used for evaluation against a range of therapeutic targets by a number of industrial and academic collaborators. Also, a statistically significant portion of the current library of 11 200 derivatives has been analyzed by mass spectrometry to confirm the presence of the expected derivatives (see note added in proof after section 15.12.4.5).

15.10 Alternate Strategies for Benzodiazepine-Based Diversity

An alternate display of functionality is possible by use of the 1,4-benzodiazepine- 2,5-dione structure. Shown on the left of Fig. 15-6 is a 1,4-benzodiazepine-2-one, discussed in this chapter. The 1,4-benzodiazepine-2,5-dione scaffolding is shown on the right.

Figure 15-6. Alternate benzodiazepine-based diversity. A 1,4-benzodiazepine-2,5-dione is shown on the right.

We have recently reported [32] a general solid-phase method for the synthesis of this class of compounds, and the construction of a library of these compounds will be reported in due course. We have also developed a silicon-based method for linkage of our 1,4-benzodiazepine derivatives [33], shown in Fig. 15-7. Upon cleavage, this strategy leaves behind no trace of the linking functionality. We have also determined that substitution of silicon for germanium increases the lability of the linker so that

Finure 15-7. A silicon-based linker for traceless solid-Dhase svnthesis.

418 IS Synthesis and Evaluation of Three I,4-Benzodiazepine Libraries

cleavage by trifluoroacetic acid is facile. These linkage methods may prove useful for the construction of libraries of aromatic compounds where no memory of the solid- phase synthesis is desired.

15.11 Conclusion

The results reported in this chapter show that the parallel, multistep, solid-phase synthesis of organic molecules is an expedient strategy for the generation of combinatorial libraries that incorporate a variety of sensitive chemical functionality. In addition, a library of only a few thousand compounds can sometimes be sufficient to identify lead compounds for further modification and improvement. We have ap- plied the principles and methods outlined in this chapter to the synthesis of a number of other therapeutically important classes of organic compounds including the prostaglandins 1341, arylacetic acids [35], and steroid derivatives 1361, and to designed recognition elements including P-turn mimetics [37] and aspartic acid pro- tease inhibitors [38].

15.12 Experimental Section

15.12.1 Reagents and General Methods

Fmoc-protected amine-derivatized pins were supplied by Chiron Mimotopes (Vic- toria, Australia). Fmoc-protected amino acids (including side-chain preprotected derivatives) [23] and 4hydroxymethylphenoxyacetic acid were purchased from Nova Biochem (San Diego, CA) or Bachem Bioscience Inc (King of Prussia, PA). All other reagents and solvents were purchased from Aldrich (Milwaukee, WI). Chemical synthesis was performed in chemically resistant polypropylene deep well microtiter plates purchased from Beckmann (Fullerton, CA), catalog No. 267006. In working with sets of pins that are not in a pin block, a peptide flask (Safe-Lab, catalog number M2570) is often useful for filtration and rinsing operations. (A peptide flask is a glass cylinder with a fine frit at the bottom and a three-way valve beneath the frit. Solvents may be forced through by nitrogen pressure at the top, and reaction mixtures may be gently agitated by bubbling in nitrogen from the bottom.) When working with sets of pin blocks, the polypropylene lids from micropipette tip boxes are convenient, chemically stable reservoirs for rinses and deprotection reactions. A set of printed documentation for the library synthesis was prepared in advance. This is used as a checklist during the construction of the library, and is a very important part of the overall process.

15.12 Experimenlal Section 4 1 S

15.12.1.1 Fmoc Deprotection of Aminomethyl Solid Support (Pins)

Fmoc-protected amines on either 1.4 pmol (“small”) or 5 pmol (“large”) pins (Chi- ron Mimotopes Ltd.) are deprotected with 20% piperidine in DMF (20 min). The pins are rinsed with DMF (4 x ), methanol (4 x ), and dried under vacuum.

15.12.1.2 2-Aminobenzophenone

As noted in the text, the support-bound 2-aminobenzophenone derivatives 8 may be prepared by two different routes. Method A : The N-Fmoc-2-aminobenzophenones 1 are prepared via a solution 2-aminobenzophenone synthesis, coupling to the HMP linker allyl ester, Fmoc protection of the aniline, allyl deprotection to provide the free acid, coupling to the solid support, and Fmoc deprotection [21,23]. Merhod B: The Stille coupling route involves the synthesis of a Bpoc-protected 2-aminoarylstannane, Mitsunobu coupling to a preactivated HMP linker, acylation onto the solid support, Stille coupling, and Bpoc deprotection [26, 311. Below we describe general solid- phase methods for 1,4-benzodiazepine library synthesis by either route using the Chiron Mimotopes pins.

15.12.2 Method A

15.12.2.1 Coupling Fmoc-Protected 2-Aminobenzophenones (1) to Pins to Give 2

Deprotected pins are added to a round-bottomed flask with DMF (0.15 ml/pin) that contains the phenoxyacetic acid 1 (0.05 M), I-hydroxybenzotriazole (0.055 M), and diisopropylcarbodiimide (0.055 M). The acylation is allowed to proceed for 12 h. The pins are rinsed with DMF (3 x), methanol (3 x), and air dried.

15.12.2.2 Fmoc Cleavage

The Fmoc protecting group of the 2-aminobenzophenones 2 is removed by treatment of the pins with 20% piperidine in DMF (20 min). The pins are rinsed with DMF, methanol (3 x), and air dried. (After this step, synthesis by this route continues in the “Amino acid fluoride acylation” section 15.12.4.1.)

420 15 Synthesis and Evaluation of Three I,4-Bentodiazepine Libraries

15.12.3 Method B

15.12.3.1 Coupling Aminoaryl Stannane Cyanomethyl Ester to Pins to Give 7

To an oven-dried Schlenk flask under nitrogen is added the active ester (5 mole equiv.), 4-dimethylaminopyridine (5 mole equiv.), diisopropylethylamine (8 mole equiv.), N-methylpyrrolidinone (minimal volume), and the deprotected pins. The reaction mixture is heated at 65°C for 12 h to give support-bound stannane 7. The pins are transferred to a peptide flask and rinsed with ethyl acetate (3 x ) and CH2Cl2 (3 x), and are then dried under vacuum. Unreacted stannane is recovered by extraction of the ethyl acetate washes with 0.2 M citric acid (3 x ) and brine, con- centration of the organic layer, and column chromatography. Multigram quantities of starting material may routinely synthesized and in general about half the material can be recovered after acylation of the solid support.

15.12.3.2 Stille Coupling Reactions

The Stille coupling reactions are performed separately for each acid chloride. To a Schlenk flask under nitrogen is added the pins, K2C03 (0.20 g/mmol support bound stannane), Pd2dba3 -CHC13 (1.0 equiv.), THF (100 ml/mmol stannane), and diisopropylethylamine (4.0 equiv.). The mixture is stirred for 3 min, at which point 20 equiv. of the appropriate acid chloride is added slowly and the reaction mixture is stirred for 1 h at room temperature. The pins are then transferred to a large reaction flask and rinsed with CH2C12 ( x 5), KCN/DMSO (to remove residual Pd), H 2 0 ( x 3), and methanol ( x 3). The protected 2-aminobenzophenones and 2-aminoacetophenones may be stored at - 20 "C.

15.12.3.3 Bpoc Cleavage

The Bpoc protecting group by treating the pins with 1070 TFA/CH2C12 (5 min), rinsing with CH2C12 (2x), and repeating the sequence once. The pins are rinsed with CH2C12 (5 x ) and methanol (3 x) to give 8, and the pins are dried under vacuum. Every pin in a given flask now has the same 2-aminoarylketone attached to it.

15.12.4 Benzodiazepine Synthesis from 2-Aminoarylketones

15.12.4.1 Amino Acid Fluoride Acylation

(Synthesis by either route is identical from this point on.) The pins may be sub- divided into vials for the acylation reaction, where the number of vials needed is

15.12 Experimental Section 42 I

equal to the number of amino acids times the number of acid chlorides or 2-aminobenzophenones used in the library construction. (Alternatively, at this step the pins may also be placed into the pin blocks so that all subsequent steps are performed in deep-well microtiter plates. We have found that reagents are conserved when reactions are performed in vials due to the smaller solvent volume needed per pin.) To each vial is added 0.2 ml/pin of a CH2C12 solution that contains 0.2 M of the appropriate Fmoc protected amino acid fluoride [22] and 0.2 M of 2,6-di- r-butyl-4-methylpyridine, providing the corresponding anilide. The coupling reactions are allowed to continue for three days to ensure complete coupling of the most hindered amino acid derivatives (valine and isoleucine). The pins are rinsed with CH,C12 (3 x), MeOH (3 x), and air dried. In order to obtain high yields in this coupling step, the Fmoc amino acid fluorides should be prepared with cyanuric fluoride, and the workup should include extraction with 1 M sodium bicarbonate (3 x ) and 1 M sodium bisulfate (3 x ) to remove any cynuric fluoride byproducts, with no further purification necessary.

15.12.4.2 Amino Acid Fmoc Cleavage and Benzodiazepine Cyclization

At this point each vial should contain one pin for each alkylating agent, with the total number of vials equal to the product of the number of amino acids and the number of 2-aminobenzophenones or acid chlorides. The pins are transferred to a 96-well microtiter plate pin block for the cyclization, alkylation and cleavage steps. The Fmoc protecting group is removed by treatment of the pin blocks with 20% piperidine in DMF (20 min). The pin blocks are rinsed with DMF, MeOH (3 x ), and air dried. The pin blocks are immersed in 5 % acetic acid in DMF or NMP at 65 "C for 12 h to provide the cyclic product 4. The pins are rinsed with DMF (2 x), MeOH (2 x ), THF (2 x ), and air-dried.

15.12.43 Benzodiazepine Alkylation

After THF and DMSO rinses, the pin blocks are immersed in a 1 : 1 (v/v) solution of 0.12 M solution of lithiated 5-phenylmethyl-2-oxazolidinone in 10% DMF in THF/DMSO and sonicated for one hour. For the sonication, the pin blocks are placed in plastic ziplock bags to maintain dryness (although we have found that 2 070

water does not adversely affect the alkylation step). The pin blocks are removed from the bags and are then immersed, without rinsing, in a 0.40 M solution of alkylating agent in DMF (prepared immediately before alkylation) and sonicated for an addi- tional three hours (again in a ziplock bag), to provide the fully functionalized, support-bound derivatives 5. The pin blocks are removed from the ziplock bag and rinsed with DMF, DMF/H,O, MeOH (air dried), and CHzClz.

422 15 Synthesis and Evaluation of Three 1,4-Bentodiazepine Libraries

15.12.4.4 Cleavage from the Support

The derivatives 5 are cleaved from the support by placing the pin blocks into microtiter plates that contain 0.25 ml/well of a solution of 85 : 10 : 5 trifluoroacetic acid/Me2S/H20 for 24 h. For benzodiazepine derivatives incorporating tryptophan, 85 : 5 : 5 : 5 trifluoroacetic acid/dimethylsulfide/H20/1,2-ethanedithiol is employed as the cleavage cocktail to prevent oxidative decomposition of the indole ring [39]. The cleavage cocktail is then removed with a Jouan RC1O.10 concentrator equipped with a microtiter-plate rotor to provide the free 1,Cbenzodiazepine derivatives 6, spatially separated in the individual wells of the microtiter plate.

15.12.4.5 Analytical Evaluation of the 1,4-Benzodiazepine Library

Evaluation of the 1Q-benzodiazepine derivatives is accomplished by reverse phase HP-LC analysis using a Rainin C,* column and a 15-100Vo gradient of methanol in water buffered with 0.1 Yo trifluoroacetic acid with UV detection at 350 nm. The compound corresponding to the major peak (usually the only peak) can be isolated and submitted for mass spectrometric analysis to verify the structure of the benzodiazepine derivative. In addition, yields for synthesis on pin supports can be determined by addition of a stock solution of fluorenone in DMF followed by reverse- phase HPLC analysis to determine the relative peak area of the lY4-benzodiazepine derivative to the fluorenone standard. The quantity of material produced per pin is then calculated from the extinction coefficients of the derivatives that were determined on material prepared on a large scale. Alternately, for the synthesis of lY4-benzodiazepines on pins producing 1.4 or 5 pmol per pin, yields can be determined by addition of an aliquot of p-xylene as an internal proton NMR standard followed by peak area integration. Finally, characterization of the unpurified benzodiazepine products may be performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) using a-cyano-4-hydroxycinnamic acid as matrix.

Note Added in Proof

A subset of the second generation library was analyzed by HPLC as described for the first generation library, and yields were found to range from 6 1 4 7 % (average 72%). In addition, 48 of the compounds (randomly selected, incorporating each of the building block derivatives at least twice) were analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) using a-cyano-4-hydroxycinnamic acid as the matrix. For 46 of the derivatives the expected molecular ion was found. For one of the undetected derivatives the hydrolytically unstable alkylating agent cyanomethylbromide was used, and the expected unalkylated derivative was found. For the other compound, no product at all was found by HPLC, although

References 423

good yields were seen for all other library members analyzed that shared at least one building block. This suggests that an aliquotting error to loss of the compound.

The 11 200 compound benzodiazepine library was also analyzed for purity, yield, and molecular ions. 'H-NMR spectroscopy of five compounds from the library showed the expected benzodiazepine as the major product in each case. Twenty derivatives were analyzed by reverse phase HPLC with UV detection at 315 nm for benzodiazepines derived from aromatic acid chlorides and 285 nm for those derived from aliphatic acid chlorides. One major product (with retention time identical to authentic material prepared on large scale) was observed in all cases, although the loading was lower than expected. To further confirm that the expected compounds had been synthesized, many of the resulting compounds were analyzed by MALDI- MS. The expected peak was found for 67 of 72 randomly selected derivatives, where each building block derivative was analyzed at least twice.

Acknowledgments

Separation of the acid chlorides into bins was performed by Steven Muskal (MDL Information Systems, Inc.) and is greatly appreciated. The technical expertise provided by Andrew Bray (Chiron Mimotopes) is gratefully acknowledged. This work was supported by the NIH, the NSF, the Arnold and Mabel Beckman Foundation, and the Burroughs Wellcome Fund. Chiron, Affymax, Tularik, Eli Lilly, and Hoff- man La Roche are also gratefully acknowledged for their support.

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results, 1995.

approaches unity.

combinatorial peptide and nonpeptide libraries || synthesis and evaluation of three...

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