At the onset of this project, we envisioned two potential pairing strategies to effect HtoT macrocyclization: (1) an intramolecular SN
Ar reaction or (2) macrolactamization (). Due to the overall success of the intramolecular SN
Ar reaction for the formation of the medium sized rings 7
in our initial study,8
we first set our sights on the application of this method to access macrolactam 12
. The synthesis of the SN
Ar substrate begins with allyl carbamate formation accomplished by treatment of amine 6
with allyl chloroformate under Schotten-Baumann conditions (). This was followed by TBS removal with TBAF and methylation of the resultant secondary alcohol with methyl iodide. This synthetic sequence led to orthogonally protected diamine 16
in 54–80% yield. The choice of DMF as a solvent and the use of excess MeI was imperative to prevent intramolecular carbamate formation. The terminal PMB and Boc protecting groups were removed simultaneously upon treatment with TFA in the presence of anisole as a cation scavenger. Amidation of the resulting crude amino alcohol was then accomplished resulting in SN
Ar substrate 14
in yields of 56–70% for the two steps.
Potential HtoT macrocyclization strategies
Synthesis of scaffold 12 via SNAr macrocyclization
The key SN
Ar macrocyclization was first attempted utilizing conditions developed in our previous studies.8,11
Treatment of 14
with NaH in THF at 0 °C led solely to the formation of oxazolidinone 18
, which could not be avoided even with careful exclusion of water and the use of KH. Heating alcohol 14
in the presence of excess CsF was also attempted, which showed no consumption of starting material by LC/MS at room temperature but ultimately led to decomposition after extended heating (> 1 day). The use of DBU in DMF17
was also unsuccessful even after extended heating. Finally, desired product 12
could be obtained using TBAF in THF/DMF (in the prescence of 4 Å molecular sieves)18
however the yields were generally low (25–45%) and highly variable. In light of the problems encountered promoting this transformation we decided to shift our efforts to a macrolactamization approach via intermediate 15
The synthesis of 15 () commenced with PMB deprotection of intermediate 6 via treatment with DDQ in buffered CH2Cl2. The primary alcohol was subsequently subjected to an intermolecular SNAr reaction with aryl fluoride 20, in the presence of TBAF and 4 Å molecular sieves in THF/DMF resulting in ether 21 in good yield (55–75%). Bis-deprotection of the Boc and t-butyl ester groups provided crude amino acid 15 which was gratifyingly converted to macrocyle 12 in good yield (83%) upon treatment with BOP-Cl in the presence of DIEA.
Synthesis of scaffold 12 via macrolactamization
In order to access the meta
-alkoxynitro macrocycle (13
), we next decided to investigate a modified synthetic route in which the intermolecular SN
Ar reaction was replaced with a Mitsunobu reaction19
to install the nitrobenzoic acid. As shown in , primary alcohol 19
was treated with phenol 22
in the presence of DIAD and PPh3
to give phenyl ether 23
in 75–85% yield. The Boc and t
-butyl ester groups were removed with TFA and the resulting amino acid (24
) cyclized in the presence of BOP-Cl to afford macrocycle 13
in 78% yield over two steps.
Synthesis of scaffold 13 via macrolactamization
With established routes to both the para- and meta-alkoxynitro scaffolds, all eight isomers of macrocycles 12 and 13 were synthesized in 5-g quantities in preparation for solid-phase library production. The key macrocyclization reaction proceeded in high yield for all eight isomers of the linear amine scaffold (). The stereochemistry of the precursors 12 and 13, along with the regiochemistry of the aryl nitro substitutent, did not appear to influence the cyclization efficiency with combined yields for the double deprotection and lactamization ranging from 75–88%.
Macrolactamization yields for all stereo- and regioisomers
In order to load the scaffolds onto solid-phase for library production, SynPhase™
functionalized with a monomethoxy Backbone Amide Linker (BAL)21,22,23
were selected. Use of the BAL linker allows for loading via an amine (in this case an aniline) without sacrificing a diversity site. Immobilization would be achieved via reductive alkylation, while subsequent N
-capping with sulfonyl chlorides, acid chlorides and isocyanates would afford the corresponding sulfonamides, amides and ureas upon cleavage with TFA. Thus, chemoselective reduction of the nitro functionality was achieved with SnCl2
to afford anilines 25
() for loading onto solid-support. Of note, reduction of the meta
-alkoxynitroaryl group proved more sluggish than the para
regioisomer requiring heating at 60 °C for full conversion. Nitro reduction proceeded smoothly for all isomers in yields ranging from 80–89%.
Reduction of aryl nitro group (only one stereoisomer shown).
With ample quantities of all stereoisomers of anilines 25
in hand, a sparse matrix design strategy was implemented to select library members to be synthesized.24
A virtual library was constructed (for each scaffold) incorporating all possible building block combinations at R1
(aniline) and R2
(secondary amine) using a master list of reagents (R1
= sulfonyl chlorides, isocyanates and acid chlorides; R2
= sulfonyl chlorides, isocyanates, acids and aldehydes).25
This provided a virtual library of 7,845 compounds per scaffold. Physicochemical property filters were then applied to eliminate building block combinations that led to products with undesired physicochemical properties.26
The following property filters were applied to yield a total of 5363 compounds: MW ≤625, ALogP −1 to 5, H-bond acceptors and donors ≤10, rotatable bonds ≤10 and TPSA ≤140. In order to increase the percentage of ‘Lipinski compliant’ products, a ‘75/25’ rule was also implemented where 75% of all library members had MW <500. A total of 496 compounds per scaffold were selected from the remaining set using chemical similarity principles, maximizing diversity but retaining near neighbors for built-in SAR. The reagents selected for library production include 22 sulfonyl chlorides, 19 isocyanates and 26 acid chlorides for aniline capping () along with 2 sulfonyl chlorides, 3 isocyanates, 21 acids and 22 aldehydes for secondary amine capping (). The same set of reagents was used for each stereo- and regioisomer, thereby maintaining the ability to generate SSAR for each building block combination. The property distribution for the selected products (7936 compounds total) is shown in and .
Building blocks for aniline capping (R1)
Building blocks for amine capping (R2)
Molecular weight and ALogP distribution for HtoT library members.
Property analysis for the HtoT library
As shown in scaffolds 25a–h
were loaded onto the BAL functionalized SynPhase L-series Lanterns via treatment with an excess of NaBH3
CN in 2% AcOH/DMF at 40 °C for 3 days. (Lanterns were equipped with radio frequency transponders to enable tracking and sorting of library members.) Scaffolds 25
contain two handles for the introduction of appendage diversity: the immobilized aniline and a protected secondary amine, both suitable for reaction with various electrophiles. The first diversity site, the aniline, was reacted with a total of 68 building blocks including isocyanates, sulfonyl chlorides and acid chlorides.27,28
The second diversity site was then revealed via removal of the Alloc group upon treatment with Pd(PPh3
in the presence of excess 1,3–dimethyl barbituric acid. This secondary amine was capped with 49 building blocks including isocyanates, sulfonyl chlorides, acids and aldehydes. This was followed by cleavage from the Lantern, which was achieved by treatment with a 1:1 solution of TFA:1,2-dichloroethane, to afford a total of 7936 products with an average yield of 8.5 μmol/Lantern. On average, yields were slightly higher for the para
-aniline derived compounds than the meta
-aniline compounds (9.1 μmol vs 8.0 μmol).29
Solid-phase library synthesis on SynPhase monomethoxy BAL Lanterns
All library products were analyzed by ultra-performance liquid chromatography, and compound purity was assessed by UV detection at 210 nm. An overview of library purity with respect to building blocks and stereochemistry is provided in . The average purity of the library was 86% with 84% of the library being >75% pure. In general all building blocks performed well during library production other than a few which are worth discussion. Reagents used for capping diversity site 1 which resulted in lower purities include ethyl sulfonyl chloride (R1 = 2) and cyclopentyl isocyanate (R1 = 25), both of which affected products predominantly for the meta- cores across most diversity site 2 building blocks. Isoxazole acid chloride (R1 = 63) led to low purity final compounds for most of the 16 cores and diversity site 2 building blocks. Overall, building blocks used to diversify the secondary amine faired better in terms of final product purity, with only one building block (oxazole-4-carbaldehyde - R2 = 47) showing reduced purities across R1 building blocks.
Figure 6 Purity analysis (UV 210 nm) for the HtoT Library. Library members are displayed as blocks of 16 isomers (8 stereoisomers X 2 regioisomers) (see legend) and reagents used for solid-phase diversification are shown on the x- and y-axes. See and (more ...)