Molecular Diversity by Olefin Cross-Metathesis on Solid Support. Generation of Libraries of Biologically Promising β-Lactam Derivatives

The application of the reagent-based diversification strategy for generation of libraries of biologically promising β-lactam derivatives is described. Key features are the versatility of the linker used and the cross-metathesis functionalization at the cleavage step. From an immobilized primary library, diversity was expanded by applying different cleavage conditions, leading to a series of cholesterol absorption inhibitor analogues together with interesting hybrid compounds through incorporation of a chalcone moiety.


Introduction
The β-lactam skeleton is an active acylating agent with numerous applications in clinical therapy [1]. Besides the well-known use as antibiotics [2], β-lactam ring has inhibitory effects on prostate specific antigen [3,4], thrombin [5], human cytomegalovirus protein [6], human leukocyte elastase [7], cysteine protease [8,9], and human fatty acid amide hydrolase [10], as well as anticancer properties [11][12][13][14][15] and neuroprotective action [16,17]. A new area in β-lactam-based drugs has been established by development of β-lactam derivatives with strong cholesterol absorption inhibitor reducing LDL concentration [18][19][20][21]. An example is the commercial drug ezetimibe, which is one of the most prescribed drugs in the US. Recently, simvastatin has been shown to provide an incremental benefit on reducing cardiovascular events in acute coronary syndrome patients, when co-administered with ezetimibe [22]. Furthermore, β-lactams have also been considered as peptidomimetic species for mimicking certain properties of proteins and, from a synthetic point of view, they are key synthons for the preparation of various heterocyclic compounds of biological importance [23][24][25].
In drug discovery related organic synthesis, one of the tools for a rapid and efficient construction of diversity-based small molecules is parallel solid-phase synthesis. Particularly, solid-phase chemistry has recently aroused interest in metal-catalyzed cross-coupling reactions since undesirable soluble homodimers can be washed away during purification providing chemoselectivity, while immobilization of one of the substrates makes its homodimerization a less favorable process due to site isolation (Scheme 1) [26]. In this regard, we and others have recognized the usefulness of solid-supported olefin cross-metathesis for generation of biologically relevant molecules [27][28][29][30][31], including a comprehensive study to understand the process [32,33]. From the viewpoint of green chemistry, using solid-phase synthetic sequences allows a significant reduction in solvent waste, since purification is performed by phase separation, avoiding chromatographic isolation of products which requires a large consumption of organic solvents [34].
We have shown that high molecular diversity in β-lactam scaffolds can be obtained using a linker cleavable by two orthogonal conditions: one by treatment with 10% TFA, and the other using a set of olefin cross-metathesis reactions. While Seeberger developed olefin linkers for solid-phase synthesis of glycosides [40], we decided to introduce a new twist using them to generate diversity during cleavage step. Thus, a series of cholesterol absorption inhibitor analogues, together with interesting β-lactam-chalcone hybrid compounds, have been generated. Our results are summarized in this extended paper [41].

Results and Discussion
Synthesis started with immobilization of 4-pentenoic acid to Wang resin by standard coupling conditions, to afford the corresponding resin 1 which, in turn, was the substrate for a solid-supported olefin cross-metathesis by reaction with 1-(chloromethyl)-4-vinylbenzene in the presence of second generation Grubbs precatalyst, according to the methodology reported by us (Scheme 3) [32]. Then, oxidation of the supported benzyl chloride 2 with DMSO/NaHCO 3 at 155 • C [42] yielded the aldehyde 3, which was characterized by 13 C gel-phase NMR (Figure 1). Aldehyde 3 was used as substrate for the synthesis of libraries of β-lactam derivatives.

Primary β-Lactam Library
For the solid-phase version of the classical Staudinger reaction between imines and ketenes [43][44][45], we first developed the synthesis of a set of immobilized imines 5a-d by treatment of the aldehyde with different amines 4a-d in refluxing benzene using a Dean-Stark trap (Scheme 4).
For the in situ generation of ketenes, we found that stable acid chlorides such as phenoxyacetyl chloride were ideal, however, when aliphatic carboxylic acids derivatives were required, the best choice was the activation of the free acid by Mukaiyama's reagent. Thus, immobilized imines 5a-d were treated with either acid chloride or carboxylic acids (6a-d) and Mukaiyama's reagent to obtain the primary β-lactam library (7aa-dd) (Scheme 4). The relative configuration of C-3 and C-4 substituents was determined taking the coupling constant between H-3 and H-4 nucleus in 1 H-NMR experiments after trifluoroacetic acid (TFA) cleavage into account. Coupling constants greater than 4 Hz were indicative of a cis configuration [46], so β-lactams 8aa and 8ab had a cis configuration while 8bb had a trans configuration ( Figure 2 and Table 1).
During Staudinger reaction, the diastereoselectivity for cis or trans configuration of the resulting β-lactams was governed by substituents on the starting building blocks (imine and ketene) [47,48]. Ketenes that were activated by heteroatoms had strong preference for formation of cis products. This was the case for β-lactams 8aa (J = 5.0 Hz) and 8ab (J = 4.4 Hz) that have been constructed from ketenes derived from dehydrohalogenation of phenoxyacetyl chloride. On the other hand, β-lactam 8bb, which was synthesized from an alkyl-ketene derivative obtained from 5-phenylvaleric acid, gave a trans configuration (J = 2.2 Hz). The presence of a heteroatom tethered to the α-position of the ketene and tended to form the cis products by conrotatory ring closure of the zwitterionic intermediate, formed through a nucleophilic attack of imine nitrogen on the electrophilic carbonyl carbon of the ketene [47]. Conversely, thermodynamically more stable trans-β-lactam 8bb was generated by isomerization of the zwitterion, when a hydrocarbon chain was tethered to the ketene. 1 H-NMR signals corresponding to protons H-3 and H-4 showed a deshielding effect when substituent of position 1 changed from N-benzyl to N-aryl, respectively (compare 8aa and 8ab in Figure 2). Geometric optimization employing a semi-empirical method such as AM1 [49] indicated a radical change in the arrangement of the aromatic ring at position 1 ( Figure 3). In the N-aryl β-lactam, H-3 and H-4 were in the same plane of the N-1 aromatic ring, while in N-benzyl β-lactam these protons were near the axis of the ring. Due to anisotropic effect, protons H-3 and H-4 in the N-aryl-β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  these protons were near the axis of the ring. Due to anisotropic effect, protons H-3 and H-4 in the N-aryl-β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)).    Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)).    β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)).    β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields.  Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)).   Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)).  Generation of this primary library was monitored by IR and 13 C gel-phase NMR. A representative example of 13 C gel-phase NMR of resin 7bd is shown in Figure 4. Signals assigned to the immobilized β-lactam are marked (*). The most representative signals were 55.1 ppm (MeO-), and 60.7/60.2 ppm corresponding to C-3 and C-4 of the β-lactam ring.

Secondary β-Lactam Libraries
Taking advantage of the multiple possibilities of cleavage, a series of secondary libraries was generated. Thus, TFA treatment of the primary library, followed by diazomethane methylation gave an eleven compounds' secondary "TFA" library ( Table 1).
The efficiency of the solid-phase synthetic strategy has proven to be excellent. Yields ranged from 26% to 60% for five synthetic steps. Analyzing the reaction outcome of the obtained trans-β-lactams (entries 3-11), no clear tendency could be observed. While yields increase from R 2 = Ph to R 2 = 4-FPh and 4-MeOPh, in the cases of R 1 = phenylpropyl and vinyl, this tendency was reverted in case of R 1 = allyl ( Figure 5).
In order to increase diversity during cleavage step, we studied the olefin cross-metathesis on the immobilized β-lactams. Thus, a new secondary library was created when β-lactams 7bb-bd, bearing a 3-phenylpropyl group at C-3 position, reacted with different olefins to afford structural diversification at C4 position (Table 2). This secondary library, called "Ru1", was present in six different alternative β-lactams (10bba-bdc) obtained by reaction of the olefins in presence of second generation Grubbs precatalyst. In accordance with our previous results in the area of olefin cross-metathesis [32], trans-crotonic acid (entries 1 and 2), a type I olefin [50] and allylbenzene (entries 3 and 4), a type II olefin, performed the cleavage process with high efficiency, while 2-bromostyrene (entries 5 and 6), a type IV olefin, provided a more poor yield.  generation Grubbs precatalyst. In accordance with our previous results in the area of olefin cross-metathesis [32], trans-crotonic acid (entries 1 and 2), a type I olefin [50] and allylbenzene (entries 3 and 4), a type II olefin, performed the cleavage process with high efficiency, while 2-bromostyrene (entries 5 and 6), a type IV olefin, provided a more poor yield.  a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bb-bd (see Table 1). c Product was esterified with diazomethane prior to purification.
Hybrid structures, obtained from combining at least two biologically significant moieties, have emerged as a novel approach in finding new chemical entities [51][52][53]. The molecular hybridization strategy has proven to be helpful in many aspects related to drug discovery, such as overcoming drug-resistance problems or improving active transport mechanisms. In particular, β-lactam-based hybrids have recently acquired importance owing to the fact that many of them exhibit very promising biological activity [54][55][56][57][58][59]. In order to further increase diversity and also obtain biologically interesting β-lactam-based hybrids, metathesis conditions, previously developed for the synthesis of chalcones, were applied for the cleavage step (Table 3) [60]. In this secondary library, called "Ru2", cleavage conditions were performed by reaction of β-lactams 7bc and 7bd with non-immobilized α,β-unsaturated ketones, being the most efficient precatalyst of Hoveyda-Grubbs carbene ruthenium complex [61][62][63]. Thus, immobilized β-lactams 7bc-bd were treated with substituted vinyl phenyl ketones 9d-f in the presence of Hoveyda-Grubbs precatalyst to yield the ]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bb-bd (see Table 1). c Product was esterified with diazomethane prior to purification.
Hybrid structures, obtained from combining at least two biologically significant moieties, have emerged as a novel approach in finding new chemical entities [51][52][53]. The molecular hybridization strategy has proven to be helpful in many aspects related to drug discovery, such as overcoming drug-resistance problems or improving active transport mechanisms. In particular, β-lactam-based hybrids have recently acquired importance owing to the fact that many of them exhibit very promising biological activity [54][55][56][57][58][59]. In order to further increase diversity and also obtain biologically interesting β-lactam-based hybrids, metathesis conditions, previously developed for the synthesis of chalcones, were applied for the cleavage step (Table 3) [60]. In this secondary library, called "Ru2", cleavage conditions were performed by reaction of β-lactams 7bc and 7bd with non-immobilized α,β-unsaturated ketones, being the most efficient precatalyst of Hoveyda-Grubbs carbene ruthenium complex [61][62][63]. Thus, immobilized β-lactams 7bc-bd were treated with substituted vinyl phenyl ketones 9d-f in the presence of Hoveyda-Grubbs precatalyst to yield the soluble compounds 10bcd-bdg which combined two recognized pharmacophoric moieties such as azetidinone and chalcone in one molecule. soluble compounds 10bcd-bdg which combined two recognized pharmacophoric moieties such as azetidinone and chalcone in one molecule. ]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bc-bd (see Table 1). c Product could not be completely purified because of impurities.
Through the analysis of the 13 C-NMR spectra of the components of obtained libraries, a clear pattern could be determined ( Figure 6). Signals corresponding to substituents of all positions are slightly affected by remaining substituents. For instance, 4-methoxy-phenyl substituent at position 1 in azetidinones 8bd, 8cd, 8dd, 10bda, 10bdb, 10bdc, 10bdd and 10bde showed similar chemical shifts regardless the remaining substituents of β-lactam. Signals of the three carbons of the β-lactam ring (C-2, C-3 and C-4) could clearly be identified, having only a remarkable and expected low-field chemical shift of C-3 in case of 3-phenoxy-cis-β-lactams (8aa and 8ab). Although there was the possibility that other signals could interfere, the patterns shown in Figure 6 were useful for a rapid detection of β-lactam ring containing compounds obtained by high throughput parallel synthesis.  Table 1). c Product could not be completely purified because of impurities.
Through the analysis of the 13 C-NMR spectra of the components of obtained libraries, a clear pattern could be determined ( Figure 6). Signals corresponding to substituents of all positions are slightly affected by remaining substituents. For instance, 4-methoxy-phenyl substituent at position 1 in azetidinones 8bd, 8cd, 8dd, 10bda, 10bdb, 10bdc, 10bdd and 10bde showed similar chemical shifts regardless the remaining substituents of β-lactam. Signals of the three carbons of the β-lactam ring (C-2, C-3 and C-4) could clearly be identified, having only a remarkable and expected low-field chemical shift of C-3 in case of 3-phenoxy-cis-β-lactams (8aa and 8ab). Although there was the possibility that other signals could interfere, the patterns shown in Figure 6 were useful for a rapid detection of β-lactam ring containing compounds obtained by high throughput parallel synthesis.

General Information
Chemical reagents were purchased from commercial suppliers and used without further purification, unless otherwise noted. Solvents were analytical grade or were purified by standard procedures prior to use. Reactions requiring inert atmosphere were carried out under high-purity dry nitrogen atmosphere. Solvents from these reactions were transferred under high-purity dry nitrogen using syringes. All reactions were monitored by thin layer chromatography (Merck, Darmstadt, Germany) performed on silica gel 60 F254 pre-coated aluminum sheets, visualized by a 254 nm UV lamp, and stained with an ethanolic solution of 4-anisaldehyde. Column flash chromatography (Merck, Darmstadt, Germany) was performed using silica gel 60 (230-400 mesh). The purity criteria were (i) the appearance of a single spot by thin layer chromatography (ii) the presence of the corresponding signals in 1 H and 13 C-NMR and (iii) the range of melting points in case of solid samples. Molecular modeling was performed with HyperChem v8.03 (Hypercube, Gainesville, FL, USA) using the AM1 method Solid-phase reactions were carried out in polypropylene cartridges equipped with a frit (Supelco, Bellefonte, PA, USA), unless reflux conditions were required. In that case, standard glassware was used. All solid-phase reaction mixtures were stirred at slowest rate. Compounds 8bb-8dd and 10bba-10bdc have been previously reported [41].

Instrumental and Physical Data
1 H-NMR spectra were recorded in a Bruker Avance spectrometer (Bruker Analytik GmbH, Karlsruhe, Germany) at 300 MHz in CDCl 3 with tetramethylsilane (TMS) as internal standard (0 ppm). 13 C-NMR spectra were recorded on the same apparatus at 75 MHz with CDCl 3 as solvent and reference (76.9 ppm). Chemical shifts (δ) were reported in ppm upfield from TMS and coupling constants (J) were expressed in Hertz. The following abbreviations were used to indicate multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and bs = broad singlet. For preparation of the samples for 13 C gel-phase NMR, 50-80 mg of resin was placed in a standard NMR tube and 0.5 mL of CDCl 3 was slowly added in order to obtain a gel, which was homogenized by sonication. Spectra were run according to the literature [64,65].
Infrared spectra were recorded on a Shimadzu FT-IR spectrometer model 8101 (Shimadzu, Tokyo, Japan). The resin samples were measured as dispersions in KBr discs, made by compression of a mixture finely powdered in an agate mortar. Approximately 3 mg sample was used in 100 mg KBr.

Conclusions
An interesting application of DOS of biologically promising compounds has been developed. A key feature was the application of the reagent-based diversification approach using a linker with different possibilities of cleavage. Starting from an immobilized primary library, diversity was expanded through many alternatives according to the reaction conditions used. A series of cholesterol absorption inhibitor analogues was obtained, as well as interesting hybrid compounds through incorporation of a chalcone group via cross-metathesis. Here, we have demonstrated the reliability of the methodology which can be suitable for generating large libraries of analogue structures.