Microwave-assisted diastereoselective two-step three-component synthesis for rapid access to drug-like libraries of substituted 3-amino- b -lactams Bioorganic & Medicinal Chemistry

Large, diverse compound libraries are an essential requisite in target-based drug development. In this work, a robust microwave-assisted synthesis for the diastereoselective generation of 3-saccharinyl-trans - b -lactams is reported. The method is optimised for combinatorial library synthesis in which deco- ration of the scaffold is varied on both the b -lactam and the saccharine moiety. Within the European Lead Factory (ELF) consortium, a library of 263 compounds was efﬁciently produced using the developed methodology.


Introduction
The identification of lead compounds in drug discovery relies heavily on high throughput screening of large collections of compounds to a biological target for a desired effect. Structural diversity in these collections is mainly limited to planar scaffolds. 3Dcomplexity, which has been proven to be an important element in drug discovery, 1 is mainly enhanced by combining a planar core with substituents. 2 While there is an increasing trend in the development of scaffolds that contain more 3D elements, the synthetic tractability of these scaffolds diminishes.
Within the European Lead Factory (ELF) consortium, [3][4][5] we aim to develop libraries of lead-like compounds that have a high molecular complexity and that are synthesized from easily accessible building blocks in a minimum number of steps. In this work, we present a scaffold that consists of a saccharine moiety that is connected to a b-lactam via its amide-nitrogen atom (4,Scheme 1A) and that can be synthesized in a single step. 6 The b-lactam core is a frequently found structural motif in antibiotics 7 targeting the cell wall synthesis by blocking the action of transpeptidases [8][9][10] and it is useful as a building block in organic chemistry. 11 Saccha-rine has, next to being an artificial sweetener, also found application in medicinal chemistry. For example as inhibitor for tumourassociated carbonic anhydrase IX and XII, 12,13 inhibitor for interferon-mediated inflammation 14 and as antidiarrheal agents by activation of the l opioid receptor. 15 Thereby, the combination of these two functionalities renders a chemical entity with great biological potential.
The perpendicular orientation of the two ring systems, together with the substituents that we aim to introduce on the sp 3 backbone of the b-lactam provide the non-planar elements in this scaffold. For functionalization, three diversification points are envisioned on the scaffold. R 1 is envisioned as proton or nitrogroup, of which the latter can be reduced to the amine 4c and subsequently be acylated to increase the diversity (4d, Scheme 1A). The two remaining diversity points are R 2 and R 3 that are introduced by choosing the appropriate imine.
Several syntheses are known for b-lactams of which the Staudinger reaction, in which an in-situ formed ketene is reacted with an imine, is the most frequently used (Scheme 1B). 16 Usually acid chlorides are used as ketene precursors in the Staudinger reaction. 17 However, these reactive species are less suitable for use in parallel synthesis. Therefore we envisioned in-situ activation of 1 to intermediate 3 using Mukaiyama's salt as a more convenient approach. 18 Although the synthesis of the proposed scaffold using the Staudinger reaction using Mukaiyama's salt has been described before, 6 the scope of the reaction has not been thoroughly examined. Moreover, due to the fact that a stepwise addition of reagents is required, this method is impractical for use in the combinatorial synthesis of a large number of analogues.
In this report we present the optimisation of the reported procedure for the combinatorial synthesis of tri-substituted b-lactams, expand the scope of the reaction and show its applicability by producing a 263 compound library using the optimised method.

Results and discussion
We started with the synthesis of saccharinylacetic acid 1a by reacting potassium saccharine 5a and bromoacetic acid 6 neat for 4 h at 110°C to afford 1a in 85% yield (Scheme 2A). Unfortunately, the potassium salt of 6-nitrosaccharine 5b was unreactive under the same conditions. We therefore resorted to a two-step synthesis in which 5b is reacted with methyl bromoacetate 7 at 70°C in DMF for 4 h after which the crude methyl ester is hydrolysed in concentrated HCl to yield 1b in 55% yield over two steps without the need for purification (Scheme 2B).
For optimisation of the Staudinger reaction, we selected saccharinyl acetic acid 1a and imine 2a as benchmark substrates. In the initial experiment we reacted 1a with Mukaiyama's salt 10 in CH 2 -Cl 2 for 8 h at reflux, followed by the addition of imine 2a and Et 3 N which was refluxed for 16 h. This afforded the desired b-lactam 11a in 85% yield with excellent diastereoselectivity (cis/trans < 5:95) according to 1 H NMR analysis of the crude reaction mixture (Table 1, entry 1). 19 Refluxing 1a, 2a, and 10 in CH 2 Cl 2 with Et 3 N as base for 16 h was equally effective since 11a was obtained in a comparable yield (82%) and diastereoselectivity (cis/trans < 5:95, entry 2).
Unfortunately, amide 12 was quantatively obtained when benzaldehyde 9 and aniline 8 were used in a one-pot reaction instead of using imine 2a, (entry 3). This can be explained by the fact that imine formation is slow compared to the acid activation and amide formation between the amine and the activated acid. Consequently, this necessitates the use of pre-formed imines in the reaction. The reaction time was reduced dramatically from 16 h to 10 min using a microwave. The reaction of 1a and 2a in a microwave at 100°C for 30 or 10 min afforded 11a in 87% and 86% yield respectively, selectively trans (entries [4][5]. With optimised conditions ( Table 1, entry 5) established, we explored the scope of the reaction with acetic acid 1a (R 1 = H) and a variety of imines 2b-2j ( Table 2). Electron withdrawing as well as electron donating aromatic rings on the R 2 -and R 3 -position afforded the products in selectively trans configuration 11b-11h in moderate yields after preparative HPLC/MS 20 (30-50%; Table 2, entries 1-7). However, the reactions of aliphatic imines 2i and 2j, derived from neohexanal and n-hexanal, resulted in mixtures of unidentified products (entries 8 and 9). While R 3 is thus limited to aromatic rings, the reaction with imines derived from isopropylamine afforded the desired products 11k-11m in reasonable yields (63-32%), but not with full diastereoselectivity (cis/trans 15:85-33:67, entries 10-12). 21 The reactions with 4-aminomethylpyridine and b-alanine derived imines 2n and 2o further emphasise the fact that N-aliphatic imines are more challenging substrates because the reaction with 2n did not afford any product and with imine 2o product 11o is obtained in only a minimal yield of 8% (entries 13 and 14). Finally, 6-nitrosaccharinyl acetic acid 2b could be used without difficulties in the reaction with imines 2c and 2b affording 11p and 11q in acceptable yields of 58 and 61% respectively as single diastereomers (entries 15 and 16).
Having established the scope of the reaction for R 2 and R 3 , we were also interested to see if it would be possible to use substituted acetic acids other than 1. Phthaloyl glycine 13 and theophylline-7-acetic acid 14 proved good substrates for the reaction and gave access to 18 and 19 respectively in good yields (89 and 65%) with full diastereoselectivity (Table 3, entries 1 and 2). Imidazol-4-acetic acid 15 and indomethacine 16 on the other hand, failed to react and resulted in mixtures of unidentified products (entries 3 and 4). To investigate whether this is caused by the less electron withdrawing properties of the aromatic systems connected to the acetic acid part, we tested the moderately electron poor p-nitrophenyl acetic acid 17 (entry 5). Indeed this acetic acid did not afford the product and consequently more electron poor acetic acids are required.
We next turned our attention to modification of the nitro group of compounds 11p and 11q (Scheme 3). Reduction to the amine and subsequent amidation using acid chlorides can further increase the diversity potential of the scaffold. Reduction of 11p and 11q was quantitatively achieved by treatment with iron in acetic acid for 3 h at room temperature to afford 23a and 23b. The acylated amines 24a and 24b were obtained in good yields when the amines 23 were treated with acetyl chloride and sodium bicarbonate in THF, illustrating the applicability of the functionalization.
With the chemistry established, we enumerated a library of 349 compounds with an average cLog P of 3.6 and average molecular weight of 502 (Chart 1). 22 For production of the library, large batches of saccharinyl acetic acids 1a (187 mmol, 45.0 g) and 1b (143 mmol, 41.6 g) were prepared using the same methods as during optimisation. Subsequently, the appropriate imines (2) were pre-formed and reacted with 1a, 10 and Et 3 N in CH 2 Cl 2 . 23 After purification of the final products by preparative LC/MS, 263 compounds were isolated with a purity over 85% (LC/MS), which corresponds to a success rate of 75%. These compounds were added to the Joint European Compound Library.

Conclusions
In conclusion, we have developed a rapid, efficient microwaveassisted synthesis of 3-amino-b-lactams, suitable for the combinatorial synthesis of 3-saccharinyl-b-lactams starting from substituted acetic acids. The resulting 3-amino-b-lactams were isolated in reasonable yields and excellent diastereoselectivity using a range of different imines and substituted acetic acids. We have shown the application of the obtained b-lactams in follow-up  chemistry, and the developed methodology was used to produce a 263 compound library.

General experimental details
All commercially available reagents and solvents were used as purchased. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 300 (75.00 MHz for 13C) using the residual solvent as internal standard (1H: d 7.26 ppm, 13C{1H}: d 77.00 ppm for CDCl3), Chemical shifts (d) are given in ppm and coupling constants (J) are quoted in hertz (Hz). Resonances are described as s (singlet), d (doublet), t (triplet), q (quartet), br (broad singlet) and m (multiplet) or combinations thereof. Electrospray Ionization (ESI) high-resolution mass spectrometry was carried out using a Waters Xevo G2 XS QTOF instrument in positive ion mode (capillary potential of 3000 V). Flash chromatography was performed on Grace Davisil Silica Gel (particle size 40-63 lm, pore diameter 60 Å) using the indicated eluent. Thin Layer Chromatography (TLC) was performed using TLC plates from Merck (SiO 2 , Kieselgel 60 F254 neutral, on aluminium with fluorescence indicator) and compounds were visualized by UV detection (254 nm) unless mentioned otherwise. Microwave reactions were performed in a Biotage Initiator + using the corresponding microwave vials.
Reversed phase preparative HPLC/MS was carried out on a Waters AutoPurification system equipped with a Waters 2998 pho- To a vial containing toluene were added aldehyde (1.0 equiv) and amine (1.0 equiv) followed by MgSO 4 . The mixture was allowed to be stirred overnight after which it was filtered and evaporated to dryness. The imines were used in the cycloaddition without further purification.
General procedure B, cycloaddition. A microwave vial was charged with dichloromethane the appropriate imine (1.0 equiv), saccharinyl acetic acid (1.0 equiv), Mukaiyama's salt (1.05 equiv) and triethylamine (2 equiv). The mixture was heated for 10 min in the microwave at 100°C. After the reaction, the mixture was concentrated in vacuo and purified on a preparative LCMS. Waters X-Bridge 30 mm Â 150 mm C 18 column; gradient, CH 3 CN:H 2 O (0.1% TFA). Fractions containing the product were automatically collected based on observed mass and UV-signal after which they were lyophilized to obtain the pure products.
General procedure C, nitro reduction. A roundbottomed flask was charged with the required nitro saccharinyl b-lactam that was dissolved in glacial acetic acid. Next iron (10 equiv) was added and the suspension was stirred for 3 h at room temperature. After completion of the reaction (TLC) the mixture was diluted with ethyl acetate and neutralized with saturated NaHCO 3 solution. The water layer was extracted three times with EtOAc. The organic layers were combined, washed with brine, dried over Na 2 SO 4 and concentrated in vacuo.
General procedure D, Library production. In an 8 mL screw cap vial a solution of amine (0.1 mmol, 1 equiv) and aldehyde (0.1 mmol, 1 equiv) in dry toluene (1 mL) was prepared. Subsequently MgSO 4 (approx. 100 mg) was added. The resulting mixtures were stirred overnight. The mixtures were filtered and the filtrate was concentrated to afford the imines. The imine was used in the next step immediately without purification. To the imines was added 2 mL of a stock solution containing the appropriate saccharinylacetic acid in CH 2 Cl 2 (0.05 mM 0.1 mmol, 1 equiv). Subsequently, 2chloro-1-methylpyridin-1-ium iodide (Mukaiyama's salt) (0.105 mmol, 1.05 equiv) and triethylamine (0.22 mmol, 2.2 equiv) were added. The resulting mixtures were overnight heated at 45°C in closed vials. The mixtures were washed with water (1 mL) after which the organic layers were evaporated. The crude mixtures were purified by preparative HPLC (acetonitrile/water mixtures containing 0.1% TFA). The product containing fractions were lyophilized to afford the pure products.

6-Nitrosaccharine acetic acid (1b)
6-Nitrosaccharine (3.5 g, 11 mmol) was dissolved in DMF (6 mL). Sodium bicarbonate (1.84 g, 21.9 mmol) was added and the mixture stirred for 30 min after which methyl bromoacetate (1.29 mL, 13.2 mmol) was slowly added to the solution that was then heated to 70°C for 4 h. The mixture was poured in water (25 mL) and the precipitate was filtered. The crude 6-nitrosaccha-rine acetic acid methyl ester was suspended in HCl (30 mL, 37% in water) and heated to 100°C for 4 h after which the hydrolysis was complete. After cooling to r.t., the mixture was diluted with water and the precipitated carboxylic acid was filtered to yield (1b) as a Bordeaux-reddish solid (1.81 g, 55%). 1