Solid-Phase Synthesis and Combinatorial Technologies

3.2 SMALL ORGANIC MOLECULES ON SOLID PHASE: SOLID-PHASE SYNTHESIS 93

process identifies a plausible synthetic route to the target starting from commercially available precursors. At present the adopted process in SPS is identical to that for classical organic synthesis in solution. The vast majority of organic reactions have been developed as homogeneous reactions in solution, and it would be preposterous to design an SPS scheme using reactions that have not been described in contemporary organic synthesis.

An extensive knowledge of organic synthesis, both theoretical and practical, is instrumental for the chemist to select a target, to design a viable synthetic route, and to individuate, from the beginning, the issues for each chemical step employed. The reader may address a number of excellent books and reviews that deal extensively with retrosynthesis and strategies in synthetic organic chemistry (2–4) to expand these relevant topics.

3.1.2 Validation of the Synthetic Route in Solution

The validation of a planned synthetic scheme in solution is the necessary starting point to design, refine, assess and carry out successfully the corresponding SPS. A synthetic scheme in solution must provide all the intermediates and the target molecule with good to excellent yields before being transferred onto SP. Sensitivity to changes of selected parameters (e.g., temperature, concentration, and solvent) should be known for the relevant reactions, and several experimental options for each step are desirable. Side reactions and side products formed at each step should be known and understood, and their dependence on reaction parameters should be determined.

The chemist must use the notions of synthetic organic chemistry to initially select the most reasonable experimental conditions, to subsequently perform the whole synthetic route, and to eventually modify the reaction parameters, if this proves necessary, to obtain a reliable, robust, clean, and high-yielding synthesis of the target molecule in solution. Even if the required synthesis is reported in the literature, it would be advisable to check carefully the experimental conditions in order to solve possible issues arising from any reaction before moving to the SPS.

3.2 SMALL ORGANIC MOLECULES ON SOLID PHASE: SOLID-PHASE SYNTHESIS

3.2.1 Design of a Solid-Phase Synthesis

The design of a successful SPS requires the proper selection of many SPS-related entities that are not present in the corresponding solution synthesis but have to be compatible with the whole SPS sequence. A brief survey of these choices is reported here and summarized in Fig. 3.2.

The starting synthon for the SPS may or may not contain suitable functionalities to be anchored directly, or via a linker, onto the solid support. Most chemical functionalities can be coupled to an existing linker due to the large number of available linkers

94 SOLID-PHASE SYNTHESIS: SMALL ORGANIC MOLECULES

			(P)
R	L	L	TARGET
	R	R	H
		reaction conditions

TARGET MOLECULE

RELEASED IN SOLUTION

R = choice of the solid support

L = choice of the SP linker (when necessary)

H = choice of the handle on the target molecule

P = choice of the protecting groups

reaction conditions: adaptation to SPS features

Figure 3.2 Crucial factors for the design of small organic molecule SPS.

(see Section 1.2). If a molecule does not contain suitable functionalities (e.g., aromatics and biphenyls), a C–H bond of the target molecule can be replaced by a traceless linker. As a general rule, the most obvious choice of handle should be favored, rather than looking for more challenging but more complex solutions. If, even after considering all the available linking strategies, no obvious choice appears, the transfer of the synthesis onto SP should be avoided.

The same principle applies to the selection of a linker. If the molecule can be safely supported directly on the grafted group of a PS resin and eventually released after the SPS, there is no need for a linker. If an assessed and/or commercially available linker is suitable for the planned SPS, this should be preferred. Linkers that can be cleaved with clean cleavage reagents (e.g., light, TFA vapors, and ammonia vapors) should be chosen when possible. Approaches such as cyclative cleavages, which also allow the clean release of the final cyclic compound into solution, should be considered. More complex linker strategies, or even studies to create a new linker tailored to the SPS needs, are justified only if the validation of a specific linker for the SPS of the target and/or the creation of a new linker moiety are the scientific rationale of the project.

The choice of protecting groups, when they are necessary for a successful SPS, is strongly influenced both by the commercial availability of protected reagents and by the linker or the bond directly linking the target molecule to the support. In fact, it is preferable to have the simultaneous release of the target molecule from the support and deprotection of all the protecting groups as the final SPS step. When a protecting group is to be removed during the SPS, it must be orthogonal to the linker used and to the SPS intermediates.Atypicalexampleisthebase-labileN-protectingFmoccarbamateinpeptide SPS using acid-labile linkers and Fmoc-compatible coupling conditions.

The solid support is normally chosen following the personal preferences of each scientist. Many supports have similar performances and often their use gives similar

3.2 SMALL ORGANIC MOLECULES ON SOLID PHASE: SOLID-PHASE SYNTHESIS 95

results. The use of PEG–PS resins is suggested when detailed on-bead NMR studies of the reaction outcome are planned, providing that the reaction conditions do not affect the stability of the PEG chains. Hydrophobic PS resins are normally used for the preparation of large batches of compounds due to their low price and when hydrophilic solvents are not used for the SPS. Macroporous supports are used for automated continuous-flow syntheses and when aqueous and/or hydrophilic conditions are necessary. The easy elimination of solvent residues from the macropores is an advantage if further water-sensitive SP steps must be performed. Macrosupports such as pins and crowns are used to simplify the handling of the support and to obtain large quantities of target molecule after cleavage. As a general rule, an SPS performed on a specific support cannot be transferred directly onto another support even when they are very similar, as the switch to another support often requires some optimization of the experimental conditions.

The reaction conditions used in solution synthesis are generally adapted to the SPS; the reaction times are usually increased, the reagents in solution are added in excess, and their concentration is increased to drive the SPS steps to completion. Typical experimental conditions are threeto fivefold excess of solution reagents and 0.2–1 M concentrations, while the optimal reaction time is evaluated via on-bead (FTIR) or off-bead reaction monitoring. If the reagents in solution are precious, a study to determine the minimum excess required to drive the reaction to completion will be necessary.

3.2.2 Validation of Solid-Phase Synthesis and Optimization of Reaction

Conditions

Once the SPS has been designed, a first attempt to validate the solution-phase reaction conditions is made incorporating the adjustments required by the introduction of the support and the linker, including the selection of appropriate washing cycles and drying procedures after each step in order to remove all the reagents in solution and evaporate the residual solvents. The presence of the resin-bound intermediates is checked by means of the most appropriate on-bead or off-bead analytical methods, and, if the expected compound is absent, appropriate modifications of the reaction parameters are made. If a specific reaction step cannot be transferred to SPS after having tried all of the sensible options, an alternative SP synthetic route may be designed by changing the parameter(s) that are deleterious to the reaction outcome. Typically, new reagents with different protecting groups are used, different linkers or supports are employed or different handles on the starting material are exploited to support it on SP. If, even after these further trials, the expected reaction product is absent, the SPS strategy has to be either radically changed, with the new synthetic route being assessed first in solution, or completely avoided.

If the SPS gives high yields of pure target molecules by simply using the conditions employed in solution, optimization efforts are not necessary (see the example in Section 3.4). Usually, however, the expected intermediates and the final target molecule are detected, but the overall synthesis requires an optimization to increase the yields and to reduce/eliminate the on-bead side products formed. First of all, the reaction kinetics are evaluated by following the reaction course either on-bead (see

96 SOLID-PHASE SYNTHESIS: SMALL ORGANIC MOLECULES

Section 1.3) or off-bead (parallel set of experiments quenched and cleaved at different times). Further experiments should also vary the amount and the concentration of the reagents in solution, the temperature, the solvent, and any other reaction parameter. The best compromise between high yields of the expected compound and the minimum amount ofon-bead sideproductsforeach reaction parametershould bedetermined,andtherelevant conditions introduced in the final experimental procedure, to provide a successful SPS scheme. This optimized SPS scheme is scaled up, when necessary, to prepare significant amounts of the target molecules for the application selected by the SP chemist.

3.3 SMALL ORGANIC MOLECULES ON SOLID PHASE: FROM SOLID-PHASE SYNTHESIS TO SYNTHETIC ORGANIC LIBRARIES

3.3.1 General Considerations

The advantages of SPS versus solution-phase synthesis for oligomers have been detailed in Chapter 2. The noniterative synthesis of small organic molecules generally requires a careful choice of linkers, reagents, and protecting groups as well as significant efforts to optimize the selected experimental conditions for each reaction step on SP. Usually SPS is not competitive with classical techniques in solution to prepare one or a few small organic molecules, a fact that is evident from the small number of SPS of nonoligomeric molecules reported until the early 1990s.

The advent of combinatorial chemistry has dramatically changed this scenario. The simultaneous synthesis of many compounds requires automated procedures both for the preparation and the purification of products, making even major efforts to optimize the SPS reaction conditions a minor factor when compared to the advantages provided by the automated synthesis and purification of large libraries of compounds. A thorough presentation of combinatorial technologies will follow in the coming chapters, and the close relationship between SPS and combinatorial technologies will be illustrated in detail and critically reviewed therein. For now, only the main steps for the conversion of an SPS strategy aimed toward a single target into the design and the validation of an SP synthetic route for a combinatorial library will be presented.

3.3.2 Decoration of a Target Molecule

When a multistep SPS has been set up successfully, the number of building blocks/reagents used during the whole process and the number of chemical functionalities present in the final target molecule are both known definitively. As outlined in Fig. 3.3 for a specific example (5), some building blocks can be replaced during the SPS by alternative compounds of the same chemical class, for example, primary amines (R1), o-hydroxyacetophenones (R2), and cyclic aminoketones (R3). The selection of these replacements for the original building block is driven by their commercial availability and by the rational design of the planned synthetic organic library (see Chapter 4 for more details). The final resin-bound molecule may then contain some functional groups that can be decorated either by modifying the chemical functionality (as for

3.3 SMALL ORGANIC MOLECULES ON SOLID PHASE 97

NO2

NH2

NH

O

NO2 O

O

N

NH Me

R2

O
NO2	O
	O
	N
NH	Me
O

OH

O

X1 N

P

R1 = 6 replacements (N-alkyl)

R1

Me

N

P

OH

Me

O

O

O

R3 N

P

R2 = 5 replacements (o-OH acetophenones)

R3 = 2 replacements (cyclic aminoketones)

X1 = reduction of the ketone, 1 replacement

X2 = alkylation/acylation, 9 replacements

O

O

	X2	N
		NH

O Me

Figure 3.3 Monomer sets R1–R3 and decoration functions X1, X2 in small organic molecule SPS.

the ketone X1 in Fig. 3.3) or by reacting them with specific classes of reagents (as for the secondary amine X2 in Fig. 3.3). After an extensive chemical assessment, the exploitation of the available building blocks R1–R3 and the decoration of functions X1–X2, as in Fig. 3.3, produced a synthetic organic library composed of 7 × 6 × 3 × 2 × 10 = 2520 compounds (R1 × R2 × R3 × X1 × X2 permutations).

The replacement of a building block with a similar reagent and the decoration of a chemical function in the final molecule may eventually produce a huge number of analogous structures. Most of the SPS strategies currently reported in the literature

98 SOLID-PHASE SYNTHESIS: SMALL ORGANIC MOLECULES

have been designed to produce a chemical library, rather than a specific target molecule. From the beginning, thus, the chemist also selects a specific SP route on the basis of its versatility with respect to the availability of similar building blocks to be used in specific SP steps, and the presence of functionalities to be decorated on the core structure of the molecules. While it may be difficult to illustrate the theory of this exploitation process, its potential should be clear through the previous example (5) and a more detailed description of another example (1), which will be reported in the next section.

3.4 AN EXAMPLE: SOLID-PHASE SYNTHESIS OF

1H-[2]PYRINDINONES

3.4.1 Target Selection, Retrosynthesis, and Validation in Solution

An excellent report from Parke-Davis (1) presented the SPS of constrained scaffolds based on the 1H-[2]pyrindinone ring shown in Fig. 3.4. Such a rigid structure, when substituted with versatile functionalities, can be used as a scaffold for the generation of constrained molecules with potential biological activities, through classical organic synthesis or via decoration to give combinatorial libraries.

Their retrosynthetic study was based around the Pauson-Khand cyclization (6), which couples an alkene, an alkyne, and a carbon monoxide source (typically dicobalt octacarbonyl) to give a cyclopentenone ring (Fig. 3.5, top). This reaction has been widely used for synthetic purposes, and some excellent reviews (7, 8) have covered its principal features and the recent improvements to its experimental conditions. This reaction, in its intramolecular version, is ideal for the assembly of the 1H-[2]pyrindi- none scaffold in two distinct versions, differing in the stereochemistry of the ring junction (Fig. 3.5, bottom). Hence, the readily available unsaturated amino acid derivatives 3.1a,b undergo intramolecular Pauson–Khand reaction to produce the two unsaturated scaffolds 3.2a,b.

Once the strategy was selected, the validation of the relevant cyclization in solution and the determination of its stereochemical outcome and yield were carried out. The synthetic scheme is reported in Fig. 3.6. The commercially available allyl (3.3) and propargylglycines (3.7) were sequentially tosylated and alkylated with propargyl and allyl bromide, respectively, to give 3.5 and 3.9. The intramolecular Pauson–Khand cyclization produced the two isomers 3.6 and 3.10, with different stereochemistries, in a stereospecific reaction (the chiral allylglycine produced 3.6 as a single enantiomer,

H

COOH

O

N

H

H

Figure 3.4 Structure of a functionalized 1H-[2]pyrindinone.

3.4 AN EXAMPLE: SOLID-PHASE SYNTHESIS OF 1H-[2]PYRINDINONES 99

R5

O

R2

R6

R3

Co (CO)

R1

+

2

8

R5

R1

R6

R4

R2

R

R4

3

Pauson-Khand cyclization

		H
	COOR	COOH
		O
	N	N
		H
	R
		3.2a
	3.1a
	COOR	COOH
		O
	N	N
	R	H
	3.1b	H
		3.2b

Figure 3.5 Retrosynthetic study: Pauson–Khand cyclization to 1H-[2]pyrindinones 3.2a,b.

while racemic propargylglycine gave the trans-enantiomeric pair 3.10a,b). The yield of each reaction step was satisfying following a single, nonoptimized experiment, and more importantly, no side products were detected after any of the reaction steps. While further investigations could have provided more information regarding the sensitivity to changes in the reaction conditions, Bolton et al. (1) switched immediately to the SPS applying a somewhat gradual approach to transfer the synthetic scheme onto SP.

3.4.2 Design and Validation of the Solid-Phase Synthesis

Some considerations regarding the design of an SPS of 1H-[2]pyrindinones, which can be made on the basis of the target structures and the solution synthetic route, are illustrated in Fig. 3.7 using the structure of 3.10a.

Structures 3.3–3.10 possess two potential handles for their support on SP. The obvious choice is the carboxylic function, which could be linked either to a chloromethyl or to a hydroxymethyl PS resin through an ester bond. The insertion of a commercially available acid-labile linker, possibly already supported onto the resin, would allow the release of the target into solution under mild conditions. Different functionalities could be released by cleaving the acid-labile linker with, for example, TFA (free acid) and amines (amides). Another possible handle is the secondary amine, which could be anchored to resin-bound carboxylates or halides and finally released as an N-acyl or N-alkyl moiety. For both handles the protection of the other functional

100 SOLID-PHASE SYNTHESIS: SMALL ORGANIC MOLECULES

	COOMe	a		COOMe		b	COOMe
3.3	NH .HCl		3.4	HN			N
	2			Ts			Ts
							3.5
	H		COOMe
c
	O
		N
			Ts
	3.6
	COOMe	a		COOMe	d		COOMe
	NH2 .HCl			HN			N
3.7			3.8	Ts			Ts
							3.9
c			COOMe				COOMe
	O		+
		N		O		N
			Ts				Ts
	3.10a H			3.10b H

a)TsCl, TEA, DCM, 0°C, 3 hrs, then rt, 2 hrs, 74% (3.4) and 84% (3.8);

b)propargyl bromide, Cs2CO3, DMF, rt, 2 hrs, 97%;

c)Co2(CO)8, DCM, rt, 2 hrs, then N-methylmorpholine-N-oxide, DCM, rt, 2 hrs, 77% (3.6) and 71% (3.10);

d)allyl bromide, Cs2CO3, DMF, rt, 2 hrs, 93%.

Figure 3.6 Validation of the selected synthetic scheme in solution: synthesis of3.6 and 3.10a,b.

HANDLE 1

COOMe

O

HANDLE 2

N

Ts

H 3.10a

HANDLE 1: ester bond with the support, linker allowed but not necessary, release as free COOH or as ester/amide

HANDLE 2: amide bond with the support, linker necessary to cleave, release as N-alkyl or N-acyl SUPPORT: no constraints related to reaction conditions

LINKER: no major constraints related to reaction conditions

REACTION MONITORING: possible, on-bead (FT-IR among others) or off-bead (stable intermediates)

Figure 3.7 Crucial factors for the design of a successful SPS of 1H-[2]pyrindinones.

3.4 AN EXAMPLE: SOLID-PHASE SYNTHESIS OF 1H-[2]PYRINDINONES 101

group is necessary. Fmoc or Boc carbamates can be considered as protecting groups for the amine, while a t-butyl ester could be used on the carboxylic group if an acid-labile linker is chosen to cleave and deprotect simultaneously the final compound. As for the support, the reaction conditions used in solution do not favor any of the most popular choices in respect to the others. Similarly, the conditions appear compatible with the presence of many linkers, including the widely used acid-labile linkers, and they should only require tuning on the basis of the different reaction kinetics in SPS. The key cyclization looks the most challenging step, even if successful Pauson– Khand reactions on SP have been reported previously (9) and subsequently (10) to the work by Bolton et al. (1). Reaction monitoring and on-bead structure determination may make use of some groups such as the α,β-unsaturated ketone, formed during the Pauson–Khand step, but the cleavage of all the stable intermediates from the resin is also a reasonable option for an off-bead yield and purity estimation.

The authors chose, sensibly, to begin by checking the Pauson–Khand outcome on SP using an advanced intermediate, 3.11 (Fig. 3.8), prepared in solution by simple hydrolysis of the methyl ester 3.9. The free carboxylic group was hooked onto the commercially available Wang–PS resin (acid-labile linker) utilizing the mixed anhy-

			O
	COOMe	COOH	O	Wang
	a	b
N	N	Ts	N
	Ts		Ts
3.9	3.11		3.12
		d	c
	COOH		O
	N		O	Wang
	Ts	O
	3.11		N
			Ts
			H
			3.13a,b

a)LiOH, THF/H2O, rt, 3 hrs, quantitative;

b)2,6-dichlorobenzoyl chloride, pyridine, Wang resin, DMF, rt, 22 hrs;

c)Co2(CO)8, DCM, rt, 2 hrs, then N-methylmorpholine-N-oxide, DCM,

rt, 2 hrs; d) TFA/DCM 1/1, rt, 1 hr, then off-bead characterization: quantitative yield (3.11), 97% (3.14), both calculated from 3.12.

O

d

O

OH

N

Ts H

3.14a,b

Figure 3.8 Intramolecular Pauson–Khand reaction on SP: chemical assessment and synthesis of 3.11 and 3.14a,b.

102 SOLID-PHASE SYNTHESIS: SMALL ORGANIC MOLECULES

dride method (11), and the yield, calculated after cleavage of a small aliquot of 3.12 with TFA (off-bead yield), was found to be almost quantitative. This resin was treated with dicobalt octacarbonyl in DCM and subsequently with NMMO, exactly as for the cyclization performed in solution. The off-bead yield obtained for 3.14a,b (from now on only the enantiomer a will be shown in the figures) was around 97%. The other diastereomeric scaffold was not prepared, a similarly high yield being assumed for the analogous sequence.

Having accomplished the key cyclization on SP, the whole synthesis was attempted following the scheme reported in Fig. 3.9. The suitable N-Fmoc amino acids were coupled to the Wang resin, deprotected, tosylated, and N-alkylated to give 3.12 and 3.21, respectively. These intermediates were cyclized, and the six-step SPS produced the two scaffolds 3.14a,b and 3.23, with calculated off-bead yields of 84 and 74%, respectively, from 3.12 and 3.21. As a comparison, the three-step synthesis in solution gave 56 and 55% yields, which are comparable to the total yield for the six-step SPS. Besides the introduction of an N-protecting group in the early steps, the synthesis was performed exactly as in solution with the one exception of the N-alkylation to give 3.12 and 3.21, which required a larger excess of reagents and an extended time (16 h rather than 2 h as in solution). Both the high yields and the simplicity of the work-up procedures for the SPS process (a single final chromatography was performed, while each solution step required extractions and chromatographies) make it more suitable than the corresponding solution-phase approach for the synthesis of 1H-[2]pyrindinones.

The high yields obtained by using similar reaction conditions to the solution-phase synthesis did not require major efforts to optimize the reaction conditions. The minor adjustments have already been described above.

3.4.3 Exploitation of the Solid-Phase Route: Toward Synthetic Organic

Libraries

The SPS route to 1H-[2]pyrindinones, shown in Fig. 3.10, can be modified not only by introducing similar building blocks (Fig. 3.10, points 1 and 2) or new reaction steps (Fig. 3.10, 3), but also by reducing (Fig. 3.10, point 4) or decorating the target scaffold on the keto group (Fig. 3.10, 5) or on the carboxylic function (Fig. 3.10, point 6). The exploitation of these options, as presented in the original work (1), will be briefly discussed here. For simplicity, only the modifications performed on the scaffold leading to 3.13a,b will be shown even if the scaffold leading to 3.22 was similarly exploited.

The use of different sulfonyl chlorides, reacted with the amine 3.16 after Fmoc deprotection (Fig. 3.10, point 1), was attempted with moderate success using a chloro-substituted aryl sulfonyl chloride (Fig. 3.11). A nitro derivative was also considered as a transient protecting group, allowing the eventual functionalization of the ring nitrogen with various alkylating reagents after cyclization, but reduction of the nitro group during the Pauson–Khand cyclization erased this option. The use of arenesulfonyl chlorides as analogues of the original p-toluenesulfonyl chloride could