Solid-Phase Synthesis and Combinatorial Technologies

7.4 ENCODING METHODS FOR SOLID-PHASE POOL LIBRARIES 313

	NO2		OMe
	R1
	N		OMe
	R1	=	Me
HOOC	Boc		OMe
	M1
			OMe
	7 representatives
	encoded by tags T1-T3		O

HOOC

O

O

R2 = H (m-substituted acid chain in respect to the acetyl)

H (p-substituted)

Me (p-substituted)

OH

R2 M2a

3 representatives

encoded by tags T4,T5

HOOC

R2 = H (m-substituted acid chain in respect to the acetyl)

				H (p-substituted)
	O			Me (p-substituted)
O
	OH
R2 M
		2b
3 representatives

encoded by tags T6,T7

O	O	O	Me
			N
				O	O	O
R3	R4
M3a		O	OMe	O
7 representatives
			NEt2		O	S
encoded by tags T8-T10

Figure 7.33 Monomer structures and tags for the SP benzopyran libraries L6–L9: M1–M3a.

and coupled with the second monomer set (steps i–l). Finally the vessels are sorted according to the individual structures and placed in a cleavage plate (step m) where pure, released library individuals are recovered after the cleavage (step n). The preparation of the same 16-member library using parallel synthesis would have required 4 (coupling with monomers A–D) + 4 (deprotections) + 16 (coupling with monomers E–H) + 16 (cleavage) = 40 reactions. Directed sorting of radiofrequencyencoded vessels required 4 (coupling with monomers A–D) + 1 (deprotection) + 4

314 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

		O		O	O		O
	R5		R6			O
			NBoc			N
		M3b		N
	3 representatives			boc	NH	N
encoded by tags T11,T12					boc
						boc
M4					H2NO2S
7 representatives					COCl			CHO
acylating/alkylating				SO2Cl		COOH
					N
agents							N
encoded by tags T13-T15				CHO
					N Cl		O
NH2			Me		OMe
R8
M5
7 representatives
encoded by tags T16-T18
			NHBoc		NHFmoc		NH
M6	HOOC			NHBoc	HOOC	N	NHPMC
						H
10 representatives				N	N		N
acylating		N
						O
				S			S
agents			O
spatially
encoded						COOH	SO2Cl
			COCl
				COCl			Me
			O
					N

Figure 7.34 Monomer structures and tags for the SP benzopyran libraries L6–L9: M3b–M6.

(coupling with monomers E–H) + 16 (cleavage) = 25 reactions. This reduction in the number of reactions performed becomes more significant when the number of chemical steps or the number of monomers increase and allows an easier and faster SPS of discretes using a modified mix-and-split technique. The apparatus to perform radio- frequency-encoded SP reactions is commercially available using either resin beads in microvessels (227), grafted microtubes (228, 229) or pins as reactors (230), and the SPS of libraries with up to several thousands of individuals with good yields and purities have been reported (231–235). The interest generated by this technique has recently stimulated improvements in the handling and sorting of large amounts of microreactors (236), in the final cleavage protocols (237), and in their direct use for

	7.4 ENCODING METHODS FOR SOLID-PHASE POOL LIBRARIES						315
	O			OH
		O	NH
O NH
O	O		O	O	N O
		O		Me
	N
	Me			7.56(L7)	NH
	7.55(L6)	NH
OMe			OMe
MeO	O	MeO		O	Me
						Me
					O
	N			N		O
						Me
	H			H
	7.57(L6)		O	7.58(L9)
		O				N
						SO2Me
			O
OMe				OMe
MeO	O	Me	MeO		O	Me
							Me
	O	Me			O	O
		O
	N		Me		N		Me
	H				H
	7.59(L6)	O			7.60(L8)	S	S

Figure 7.35 Structures of six characterized individuals 7.55–7.60 from L6–L9.

biological screening (238). We can easily foresee a steady and copious flow of reports of radiofrequency-encoded SP libraries in the future, especially considering the reasonable cost of the necessary equipment, which is already present in most laboratories that perform combinatorial synthesis of chemical libraries.

7.4.4 An Example: Synthesis of a Tyrphostin Radiofrequence Encoded

Library

Recently Shi et al. (239) reported the synthesis of a 432-member focused library L10 as a source of tyrosine kinase inhibitors using benzylidene malononitriles, or tyrphostins, as structural motifs to design the library. Both the general tyrphostin structure (7.61) and an example of an active compound on a specific tyrosine kinase (7.62) are reported in Fig. 7.37.

The reactors selected were 432 aminomethylated MicroTubes 7.63, which are polystyrene-grafted polypropylene tubes with a ≈50 M loading capacity per reactor, that encapsulate a preencoded radiofrequency tag (Fig. 7.38). The tubes were reacted with the Fmoc-protected, acid-labile Knorr linker (step a), the residual amine functions

316 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

		a,b		a,c			a,e
					a,d
	A	A	B	B		C	C	D	D
	E	F	E	F		E	F	E	F
	A	A	B	B		C	C	D	D
	G	H	G	H		G	H	G	H
					f,g
A A		A A B B		B B C C			C C D D		D	D
E	F	G H E	F	G H E		F	G H E	F	G	H
	h,i			h,j	h,k			h,l
	A	B	A	B		A	B	A	B
	E	E	F	F		G	G	H	H
	C	D	C	D		C	D	C	D
	E	E	F	F		G	G	H	H
					m,n
AE	AF	AG AH BE	BF	BG	BH CE	CF	CG CH DE	DF	DG	DH

16-member discrete library of released compounds

a: sorting according to first monomer addition; b-e, i-l: coupling of resin with monomer: A (b), B (c), C (d), D (e), E (i), F (j), G (k), H (l); f: mix in one pool; g: deprotection; h: sorting according to second monomer addition; m: sorting according to individual structures; n: cleavage.

Figure 7.36 Radiofrequency encoding: directed sorting for a hypothetical 16-member discrete library.

were capped (step b), and the Fmoc group was removed (step c) to give 7.64 with a ≈50 M loading (Fmoc reading). The above reactions were performed in a single flask, while the coupling with M1 (18 aromatic aldehydes) required prior sorting of the preencoded tubes (step d) and their partitioning into 18 flasks, where each aldehyde

7.4 ENCODING METHODS FOR SOLID-PHASE POOL LIBRARIES 317
R2	CN	HO	CN
			H
	R1	HO	N
(R3O)n
			O
7.61	O	7.62

Figure 7.37 General structure of tyrphostins (7.61) and of an active compound on a tyrosine kinase (7.62).

was coupled to 24 tubes (step e), and the corresponding imines were reduced (step f) to give secondary amines 7.65. All the reactors were then pooled (step g) and coupled with cyanoacetic acid using multiple cycles (step h) to give the amides 7.66. The tubes were then sorted according to M2 (eight aromatic aldehydes bearing a phenol group,

RFTag

CHO

NH2

a-d

H

NH2

e-g

H

H

N

+

N

N

L

L

R1

7.63

7.64

R1

7.65

M1

PS-grafted

tubes

18 sorted pools

18 representatives

24 tubes/pool

CN

CHO

(OH)n

O

R2

CN

H

O

h,i

N

+

j,k

N

(OH)n

H

+

L

R1

N

R2

N

7.66

L

R

M2

7.67

1

8 sorted pools

54 tubes/pool

8 representatives

3 sorted pools

144 tubes/pool

(OCOR3)n

(OCOR3)n

CN

CN

R2

R2

O

COCl

O

+ R

l

m,n

3

M3

H

L10

NH

N

N

R1

3 representatives

L

R1

432 discretes

7.68

a: Knorr linker, PyBOP, DIPEA, DCM; b: Ac2O, DIPEA; c: 20% piperidine/DMF; d: sorting according to M1; e: coupling with M1; f: NaCNBH3, AcOH; g: mix in one pool; h: cyanoacetic acid,

DIC, DMF, three cycles; i: sorting according to M2; j: coupling with M2; k: sorting according to M3; l: coupling with M3; m: sorting as individual compounds; n: 4% TFA/benzene.

L = Knorr linker

Figure 7.38 SP synthesis of a radiofrequency encoded, 432-member discrete tyrphostininspired library L10.

318 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

step i), and the aldol condensation was performed in eight flasks, each containing 54 tubes (step j), to give the unsaturated nitriles 7.67. Finally, the tubes were again sorted, this time according to M3 (two acyl chlorides and an empty position to keep the free phenol, step k), and coupled to give the final supported esters/phenols 7.68 (step l). These were sorted and identified (step m), then accordingly arranged in 432 positions of several microplates and cleaved (step n, Fig. 7.38) to give the library L10 as 432 discretes with good yields and purities determined by TLC of all samples and by MS and NMR of 5% of library compounds selected randomly (24 samples).

The power of this encoding method to prepare discrete libraries is again summarized by the number of chemical reactions performed. If the library had been prepared by parallel synthesis, a total of 1 (step a) + 1 (step b) + 1 (step c) + 18 (step e) + 18 (step f) + 18 (step h) + 144 (step j) + 288 (step l) + 432 (step n) = 921 reactions would have been necessary. Directed sorting allowed the number to be limited to 1 (step a) + 1 (step b) + 1 (step c) + 18 (step e) + 18 (step f) + 1 (step h) + 8 (step j) + 2 (step l) + 432 (step n) = 482 reactions, while ensuring the same quality of the final discrete products.

7.5 NEW TRENDS IN SOLID-PHASE POOL LIBRARIES

7.5.1 Bead-Based Libraries: High-Throughput Synthesis

Many factors have recently contributed to a general decrease of interest for large, primary SP pool libraries. These include the increased throughput of solution and SP discrete libraries, the advent of computational tools to create virtual libraries and to rationally select smaller subsets to be prepared, and the assumed lower quality of large SP pool libraries, which originates from their more difficult analytical characterization. Despite all these factors, some recent examples of bead-based pool libraries have clearly shown how a rigorous, integrated approach may produce high-quality, very large primary or biased-targeted libraries to be tested using HTS. The amount of information generated by these libraries, together with the moderate amount of efforts necessary, should convince even the more conservative chemist of the huge potential embedded into this library format and that a rigorous chemical approach is the gateway to successful SP pool libraries.

We will present here a very recent example by Tan et al. (196, 240), who are heavily involved in the so-called chemical genetic approach, (see Section 9.1.4) where large numbers of compounds must be routinely screened to identify small-molecule ligands/inhibitors, which are able to influence as many gene products as possible. This group developed some miniaturized, cell-based HTS (241–244), which allow testing of bead-based libraries and detection of active, cell-permeable compounds that interact with proteins (242), or even compounds that influence posttranslational modifications of gene products (243). These miniaturized assays require large collections of meaningful small molecules to be tested, and the authors decided to use encoded (189), bead-based libraries prepared by mix-and-split methods. They introduced some limitations upfront, such as water-compatible Tentagel supports and a photolabile linker

7.5 NEW TRENDS IN SOLID-PHASE POOL LIBRARIES 319

(245) which are limiting the choice among many organic reaction conditions, and also decided to start from structures similar to known natural products and to combinatorialize them to obtain millions of small organic compounds. Even more importantly, they decided to be extremely rigorous in assessing the purity of such a large SP pool library.

The authors chose as a template to inspire their large bead-based library synthesis the compound 7.69 (Fig. 7.39) (246, 247). This polycyclic scaffold is interesting as such, representing a constrained, highly functionalized small organic molecule, but it can also be considered as a highly flexible, geometrically and stereochemically pure scaffold where to introduce chemical diversity by mild chemical transformations without the need of protection/deprotection steps. A reasonable retrosynthetic scheme was designed to provide the 7.69-inspired SP scaffold 7.70 (Fig. 7.39). This scaffold allows a priori four primary diversifications (Fig. 7.39, full arrows): 1) nucleophilic addition to the epoxide, 2) nucleophilic addition to the lactone, 3) decoration of the R1 substituent (either through different nitrones, or using functionalizable nitrones), and 4) reductive cleavage of the N-O isoxazoline bond. Transformations 1, 2 and 4, if successfully assessed, would open secondary diversifications (Fig. 7.39, dashed arrows): 1′) decoration of the hydroxyl function originated from epoxide opening; 2′) decoration of the hydroxyl function originated from lactone opening; 3′) decoration

O	H
	N			nitrone
O		O		decoration		amine
						decoration reductive
H	H	H				N-O bond
						cleavage
	7.69		lactone	O H	R1	alcohol
			opening		N	decoration
		alcohol		O	O
						H
		decoration		H		N
						L
			alcohol	O		O
		decoration				7.70
				nucleophilic
				epoxide
				opening
			HO	COOH		O
	HO	COOH				HOOC N
						R1
						7.72
	O		O
	(+)-7.71		(-)-7.71

Figure 7.39 Bead-based libraries: structures of a natural product-biased scaffold 7.69 and of its adapted SP version 7.70 for the synthesis of a large bead-based library.

320 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

of the hydroxyl function originated from N-O cleavage; 3′′) decoration of the amine function originated from N-O cleavage.

Having planned to synthesize a large library and to use a large number of monomers, the authors were particularly strict in the SP chemical assessment for each of the above transformations. While all of them produced the expected compounds, even after an extensive optimization of the reaction conditions several of them either resulted limited to only a few substrates (not enough flexible) or did not provide the reaction products with enough purity (cut-off >90% after photocleavage of the linker). The reader is invited to read carefully the ref. 240, which is an ideal example of rigorousness in assessing combinatorial-friendly SP transformations, to access all the details of this feasibility study. The result of this assessment focused the authors’ efforts on three high quality transformations (boxed, Fig. 7.39): lactone opening with amines, esterification of the resulting OH and decoration of R1 via the use of bis-functionalized benzylnitrones.

The two epoxide enantiomers (+) and (–) 7.71 were selected as chemical starting points for library generation: Their synthesis from shikimic acid with reasonable overall yields was already known (248, 249), and they had a carboxylic acid handle for SPS. The other key intermediates were the three benzylnitrone carboxylic acids 7.72a–c, which were prepared from the corresponding benzyl alcohols (250, 251) (Fig. 7.40). The two epoxycyclohexenols were supported onto a PEG-based resin, loaded with a photolabile linker, to give resin-bound 7.73a,b (from now on only one enantiomer will be shown in the figures, but the synthetic pathway was continued with both

					HO	COOH
				3 steps	O
					(-)-7.71
HO	COOH	HO		COOMe
HO		HO
	OH		OH		HO	COOH
shikimic acid				3 steps
					O
					(+)-7.71
	X		O	X
	3 steps	HOOC	N

HO

7.72

X = o- (7.72a), m- (7.72b) and p-I (7.72c)

Figure 7.40 Solution synthesis of key synthons (+)-7.71, (–)-7.71 and 7.72a–c.

7.5 NEW TRENDS IN SOLID-PHASE POOL LIBRARIES 321

enantiomers of 7.71). These two compounds were condensed with nitrones 7.72a–c, producing six resin-bound tetracyclic benzyl iodides 7.74 (Fig. 7.41).

The authors selected a multistep synthetic pathway in accordance with the findings from chemistry assessment employing (a) an alkyne coupling with the iodoaromatic function, (b) the opening of the lactone ring with an amine, and (c) the esterification of the generated hydroxyl with carboxylic acids. They started from the six resin-bound compounds 7.74a–c and 7.75a–c (containing an ε-aminocaproic acid spacer, Fig. 7.42) and prepared six final, released compounds 7.82a–c and 7.83a–c using different monomers, cleaving and isolating a portion of each intermediate 7.76–7.79 in solution (Fig. 7.42). A complete off-bead analytical characterization of intermediates and final compounds (NMR, MS, TLC, HPLC) confirmed an 80–90% purity of photocleaved compounds, with good yields from the six-step SP sequence [from the loading of

(+)-7.71 onto the two resins] for all the compounds.

These encouraging results employing mild chemical conditions prompted the authors to perform a monomer rehearsal: The scheme of this crucial operation is reported in Fig. 7.43. Scaffold 7.74a was selected and reacted in 50 different vials with 50 alkynes according to the Sonogashira–Castro alkyne coupling; 23 monomers produced, after cleavage, the expected coupling product with >90% yields and purity (HPLC, HPLC-MS, sometimes TLC and FAB-MS) and were selected, together with seven less optimal monomers (>70% yields, high purity), to give a first monomer set of 30 rehearsed alkynes. A large amount of scaffold 7.76a was then prepared by coupling t-butylacetylene with 7.74a, and this intermediate was reacted with 87 primary amines to give 54 high-performing monomers and 8 less optimal ones, for a

		HO		COOH	O
				HO	L
					N
L NH	+		O	a	H
	2		(+)-7.71	O	7.73a,b
			or		+ 7.72a-c
			(-)-7.71
					I
			O	H
		b		N
			O	O
				H
			H	N
				L

O

O

7.74a-c (from (+)-7.71) 7.74d-f (from (-)-7.71)

a: PyBOP, DIPEA, NMP, rt; b: HATU, DIPEA, DMAP, DCM, rt.

Figure 7.41 SP synthesis of key intermediates 7.74a–f.

322 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

R1

I

O

H

O

H

N

N

O

O

H

O

O

H

N

L

R1 a

H

N L

S

+

H

O

S

O 7.76a-c

O

O

7.77a-c

L = photolinker

S = absent (7.74a-c)

+

NH2

S = spacer (7.75a-c)

R1

R

R1

2

R2 NH

R2

NH

O

N

+

COOH

O

N

b

O

H

R3

R3

O

O

H

HO

L

L

N

c

N

S

S

O

O

O

O

O

7.80a-c

7.78a-c

7.79a-c

7.81a-c

EtO

OEt

OMe

OMe

d

NH

NH

O

N

O

and

O

N

O

O

O

O

H

NH2

N

NH2

O

O

O

O

7.83b

7.82b

O

O

7.81a R1 = t-Bu (o-substituted); R2 = p-MeOBenzyl; R3 = i-Pr 7.81c R1 = n-Pr (p-substituted); R2 = p-MeOPhenethyl; R3 = Et

7.83a R1 = benzyl (o-substituted); R2 = cyclobutyl; R3 = p-MeOBenzyl

7.83c R1 = p-ClPhenyl (p-substituted); R2 = o-MeOBenzyl; R3 = i-Pr

a:CuI, (PPh3)2PdCl2, DIPEA, DMF, rt, 15-45'; b: 2-HOPyridine, THF, rt, 12-16 hrs;

c:DIC, DIPEA, DMAP, DCM, rt, 12-16 hrs; d: cleavage.

Figure 7.42 SP chemistry assessment for the large, natural products–biased library L12: compounds 7.82a–c and 7.83a–c.

second monomer set of 62 rehearsed primary amines. A similar procedure was repeated with resin-bound 7.78a, obtained by reaction of 7.76a with p-methoxybenzyl amine, and 98 carboxylic acids were tested to eventually produce a third rehearsed monomer set made of 62 carboxylic acids (44 high performing, 18 less than optimal, Fig. 7.43).