Isonitrile-Based Multicomponent Synthesis of β-Amino Boronic Acids as β-Lactamase Inhibitors

Abstract

The application of various isonitrile-based multicomponent reactions to protected (2-oxoethyl)boronic acid (as the carbonyl component) is described. The Ugi reaction, both in the four components and in the four centers–three components versions, and the van Leusen reaction, proved effective at providing small libraries of MIDA-protected β-aminoboronic acids. The corresponding free β-aminoboronic acids, quantitatively recovered through basic mild deprotection, were found to be quite stable and were fully characterized, including by ¹¹B-NMR spectroscopy. Single-crystal X-ray diffraction analysis, applied both to a MIDA-protected and a free β-aminoboronic acid derivative, provided evidence for different conformations in the solid-state. Finally, the antimicrobial activities of selected compounds were evaluated by measuring their minimal inhibitory concentration (MIC) values, and the binding mode of the most promising derivative on OXA-23 class D β-lactamase was predicted by a molecular modeling study.

1. Introduction

The recent interest in using boron in medicinal chemistry is due to the reliable fact that boron is a versatile atom, potentially not toxic, and thus able to play an important role in drug design [1].

The presence of an empty p-orbital promotes the easily conversion of the boron center from neutral trigonal planar sp² to tetrahedral sp³ hybridization, imparting peculiar chemical behaviors to boron-containing compounds. Indeed, and thanks to the empty p-orbital, boronic acids can form tetracoordinate “ate” complexes by interacting with nucleophilic groups, including the side chains of some amino acids such as hydroxyl of serine and threonine. Further, tricoordinate boron can display several types of interaction with active site nucleophiles, leading to various and predictable coordination modes upon biological targets [2].

In the last few years, examples of new bioactive compounds containing boron have been widely reported, mainly in the anti-infective [3] and anticancer fields [4]. Some of them have been developed as drugs and recently received FDA approval, such as bortezomib, for the treatment of multiple myeloma, and vaborbactam, a potent inhibitor of β-lactamase (BL) enzymes, approved for use in combination with meropenem.

Thanks to their ability to form a tetrahedral adduct with the catalytic serine, boronic acid-containing compounds have been widely used in the design of β-lactamase transition state inhibitors [5,6]. Unlike commercially available β-lactam-based BL inhibitors (e.g., clavulanate, sulbactam, and tazobactam), which are effective primarily against class A BL, boronic acid-containing compounds have been demonstrated to strongly inhibit other classes of BLs, such as the clinically-relevant class B metallo-carbapenemases, class C enzymes, and OXA-type (class D) carbapenemases [7].

Since the rapid rise of antimicrobial resistance is currently one of the greatest medical challenges, the development of novel BL inhibitors is of utmost importance, particularly for the treatment of challenging infections caused by β-lactamase-producing Gram-negative species, which are more and more commonly resistant to carbapenems and exhibiting a multi-drug or even pan-drug resistance phenotype.

Looking at their chemical structures, most relevant boron-containing drugs, including bortezomib and vaborbactam, are characterized by the presence of a α-aminoboronic acid moiety. Such fragments mimic the naturally occurring amino acids and can act as their bioisosteres, acquiring huge potential as a privileged motif in peptidomimetic chemistry. A variety of valuable protocols have been established for the synthesis of α-aminoboronic acid derivatives [8], and for exploiting various boron-protecting groups aimed at controlling the high reactivity of the C-B bond and preventing its linkage [9].

Taking into account the pronounced tendency towards degradation of α-aminoboronic acids and aiming to expand the chemical diversity of aminoboronic acids-based compounds, Professor Yudin and coworkers recently explored the reactivity of boron-containing compounds, making use of the versatile N-methyliminodiacetyl(MIDA) boron-protecting group [10,11]. In particular, they provided access to novel boron and nitrogen-containing building blocks, developing the new class of β-aminoboronic acid derivatives, characterized by the presence of an additional C-atom between the boronic acid and amino moieties. Activity-based protein profiling of such homologues demonstrated their suitability as chemical probes of human enzymes, paving the way for their possible development as drugs [12,13].

Synthetic methods for preparation of both α and β-aminoboronic acids have been reviewed quite recently [14]. Said review attested to the rapid development in the field, with most of the protocols having been reported in the literature over the last decade. Even if major advances regarding the synthetic accessibility of such derivatives have been made, most of the reported strategies still rely on multi-step sequences, though there is a single example of a multicomponent strategy [15]. Owing to their ability to construct highly functionalized molecular scaffolds from simple precursors in a single step, multicomponent reactions (MCRs) can be considered excellent tools for diversity-oriented synthesis applications in the drug discovery field [16].

As part of our ongoing interest in isonitrile-based MCR applied to drug discovery projects [17,18,19,20,21,22,23], we herein report on the application of the Ugi reaction, both in the four components (4CR) and in the four centers-three components (4C–3CR) versions, for the rapid construction of two small libraries of β-aminoboronic acids. We also demonstrate the suitability of the isonitrile-based van Leusen reaction for the preparation of a MIDA-protected methylboronic acid, bearing a substituted imidazole ring (Scheme 1).

By means of simple and rapid protocols, 20 compounds have been prepared, showing a wide degree of appendage diversity, finely modulated with regard to stereoelectronic properties. A selection of such compounds was also tested for the ability to potentiate the activity of β-lactam antibiotics (a series of isolates produced various BL enzymes) to get early insights into and preliminarily information on their potential biological activities. Finally, the putative binding mode of the most promising compound on OXA-23 class D β-lactamase was predicted by a molecular modeling approach.

2. Results and Discussion

2.1. Synthesis

We based our investigation on MIDA-protected α-boryl aldehyde 1, employing p-Br-aniline 2a, t-butyl isocyanide 3a and trans-cinnamic acid 4a for the initial screening of the Ugi-4CR conditions. Even though MCRs run best in methanol and trifluoroethanol, we looked at aprotic solvents, such as acetonitrile, dioxane, and toluene, in order to preserve the MIDA-protecting group. Acetonitrile proved to be the best choice, affording product 5a in 46% yield, when the reaction was conducted at 60 °C, for 24 h. A curcumin-based TLC colorimetric method has been successfully employed for the qualitative detection of boron-containing derivatives, during the progress of the reaction [24]. A definite increase of the yield (69%) could be obtained by adding the isocyanide in portions over three h while keeping the reaction at room temperature, and then heating at 60 °C for the remaining time. In this way, the competitive Passerini reaction was completely discouraged. Finally, the addition of the weak Broensted acid ammonium chloride proved to be beneficial, likely through promotion of imine formation and activation to nucleophilic attack from isocyanide [25]. The optimized conditions which facilitated achieving a 74% satisfactory yield for 5a are reported in Scheme 2. Under such conditions, sixteen MIDA-protected β-aminoboronic acid derivatives (5a–p) have been easily obtained, changing the amine (2a–d), isocyanide (3a–e), and carboxylic acid (4a–c) components.

Linear and branched alkyl isocyanides, and benzyl isocyanide and isocyano acetate, were successfully employed. Besides p-Br-aniline and trans-cinnamic acid, benzyl amines, lipophilic alkyl amines, benzoic acids, and long chain aliphatic acids proved to be suitable, respectively, as amine and carboxylic acid components, affording the desired MIDA-protected compounds 5a–p in moderate, but satisfactory yields. In addition to HR-MS, ¹H and ¹³C-NMR characterizations for all compounds, the reference compound 5a was subjected to ¹¹B-NMR and single-crystal X-ray diffraction.

As shown in Figure 1, in the solid-state, compound 5a lacks strong hydrogen bond donors, apart from the amide NH that is involved in an intramolecular contact with the tertiary amide carbonyl. Accordingly, the molecule assumes an extended conformation, with cumbersome phenyl rings mutually orthogonal to each other and the two five-membered rings almost perfectly flat.

In order to provide unprotected boronic acids for biological evaluation, reference compound 5a was subjected to 3M HCl in acetonitrile, for removal of MIDA according to literature procedure. The corresponding β-aminoboronic acid 6a could be isolated in a good yield after reverse-phase chromatographic purification, which allowed removing a small quantity of tertiary amide hydrolysis byproduct. Aiming at a cleaner protocol, also suitable for acid-labile compounds, an alternative, basic MIDA-deprotection was applied, treating compound 5a with excess of sodium carbonate in dry methanol, at room temperature for four h. Compound 6a was recovered quantitatively, without the need for purification. Said protocol was extended to all MIDA-protected compounds and afforded the desired β-aminoboronic acids 6 in excellent yields, also in the presence of susceptible methyl acetate groups (compounds 6o–p).

The free boronic acids 6a–p proved to be quite stable towards the common protodeborylation reaction [27] and were fully characterized by means of HR-MS, ¹H, ¹³C, and ¹¹B-NMR. A single-crystal X-ray diffraction analysis was performed on 6d, as reported in Figure 2. Unlike 5a, the free β-aminoboronic acid 6d contains strong hydrogen bond donors B-OH, which are all involved in extended ribbons networks in the solid state.

Aiming to expand the diversity of peptidomimetic β-aminoboronic acids, including the privileged β-lactam ring in the chemical structure, a 4C–3CR version of the Ugi reaction was investigated (Scheme 3).

Keeping α-boryl aldehyde 1 and t-butyl isocyanide 3a as fixed components, β-alanine 7a, or alternatively, enantiomerically pure β-amino acids (S)-3-amino-3-phenylpropanoic acid 7b or (S)-3-amino-4-methoxy-4-oxobutanoic acid 7c, were employed in the reaction. The reaction conditions optimized for the Ugi-4CR proved effective for this three-component version also, affording compounds 8a–c in acceptable yields. MIDA-deprotection with excess of sodium carbonate in dry methanol afforded, quantitatively, the corresponding β-lactam-based β-amino boronic acids 9a–c. The chirality of β-amino acids 7b and 7c did not exert any asymmetric induction on the reaction pathway, so compounds 8b and 8c (and consequently 9b and 9c) were obtained as inseparable mixtures of enantiomerically pure β-lactams diastereoisomers and characterized as such. A unique signal at 31 ppm could be detected in the ¹¹B-NMR spectrum, for both diastereoisomers of compounds 9b and 9c.

Finally, the possible use of α-boryl aldehyde 1, together with a primary amine and toluenesulfonylmethyl isocyanide (TosMIC) in a three-component reaction, was briefly explored. Cyclohexylmethanamine was chosen as the amine component and subjected to a precondensation time of two h at 50 °C in DMF with aldehyde 1. After that, TosMIC and K₂CO₃ were added in two portions and the reaction was left at 50 °C overnight. The desired MIDA-protected (imidazolyl)methyl boronic acid 10 could be obtained, establishing the first application of the van Leusen reaction to the synthesis of boron-containing imidazoles (Scheme 4). Given the modest yield (32%) achieved by then, and after a brief screening of reaction conditions, MIDA-deprotection was not carried out, reserving it for further optimizations studies.

2.2. Biological Evaluation

Some of the synthesized compounds were subjected to a preliminary characterizations of their biological activities, in which their potential synergistic activities with β-lactam antibiotics were evaluated in a set bacterial strains producing various BLs, including both laboratory strains and clinical isolates (

Table 1. In vitro antimicrobial susceptibility testing performed on both isogenic Escherichia coli laboratory strains and clinical isolates producing various β-lactamases (BLs) in the presence of fixed concentrations (16 or 32 µg/mL) of the selected compounds (AMP, ampicillin; IPM, imipenem; FEP, cefepime).

Strain	BL Produced	MIC (µg/mL) a
Isogenic strains b		AMP	AMP + 6m	AMP + 6b	AMP + 6k	AMP + 6e
E. coli DH5α(pLBII-CTX-M-15)	CTX-M-15	512	512	512	512	512
E. coli DH5α(pLBII-KPC-2)	KPC-2	256	256	256	256	256
E. coli DH5α(pLBII-AmpC-EC)	Enterobacter cloacae AmpC	128	128	128	128	128
E. coli DH5α(pLBII-CMY-2)	CMY-2	128	128	128	128	64
E. coli DH5α(pLBII-OXA-10)	OXA-10	512	512	512	512	512
E. coli DH5α(pLBII-OXA-23)	OXA-23	512	256	256	256	128
E. coli DH5α(pLBII-OXA-40)	OXA-24/40	256	128	128	128	128
E. coli DH5α(pLBII-OXA-40)	OXA-48	64	64	64	64	64
Clinical isolates c		IPM	IPM + 6m	IPM + 6b	IPM + 6k	IPM + 6e
E. coli SI-44	KPC-3, CTX-M-15, TEM-1	4	4	2	4	2
K. pneumoniae SI-109	KPC-3, SHV, TEM-1	16	16	16	16	16
		FEP	FEP + 6m	FEP + 6b	FEP + 6k	FEP + 6e
E. coli 26sm02	CMY-2	2	1	1	2	2

^a Minimal Inhibitory Concentration (MIC) values determined in triplicate. ^b Compounds tested at a fixed concentration of 16 µg/mL. ^c Compounds tested at a fixed concentration of 32 µg/mL.

). As a result, and when tested at a fixed concentration of 16 µg/mL, no or limited potentiation of the antibiotic activity could be observed with BL-producing laboratory strains. Interestingly, compound 6e showed a four-fold potentiation of the activity of ampicillin when tested on a strain producing the OXA-23 carbapenemase. A modest two-fold potentiation was also observed with some compounds (tested at 32 µg/mL) when tested on clinical isolates. These data overall indicate that the compounds might still lack sufficient inhibitory potency on their β-lactamase target or that they might not be able to accumulate at significant concentrations in the bacterial periplasm (due to, e.g., outer membrane impermeability or active efflux). However, these data are overall encouraging, and indicate that the present family of compounds, and future derivatives, should be further investigated.

2.3. Molecular Modeling Studies

Hypothesizing that compound 6e could exert its antimicrobial activity by the covalent inhibition of the OXA-23 β-lactamase, computational studies were accomplished; we aimed to acquire atomistic details on the compound 6e/target reciprocal interaction. This study could be useful for the rational design of new and more potent β-lactamase inhibitors.

The OXA-23 molecular model was created following the computational procedure reported in the Materials and Methods section. Since the racemate of 6e was biologically evaluated, covalent docking of both enantiomers of compound 6e was initially performed. Hypothesizing that our compounds could act as competitive ligands, the Oγ atom of the catalytic residue Ser79 of OXA-23 was used as an anchor point for the covalent docking of both enantiomers of 6e; the CovDock algorithm, available on the Maestro modeling suite [29], was used for this calculation. The results suggested that the (R)-6e enantiomer could be the eutomer, due to a score 0.7 higher than the other enantiomer (−3.3 vs. −2.6, respectively). Then, further investigations were accomplished only on the (R)-6e/Oxa-23 complex, by our undertaking of 250 ns of molecular dynamics (MD) simulations by means of the DESMOND algorithm [30]. By these simulations, the ligand/enzyme reciprocal adaptation was allowed, and finally, the “simulation interactions diagram” tool of Maestro permitted us to gain insights into the putative binding mode of compound (R)-6e, by processing the trajectory frames acquired during the MD simulation run. As shown in Figure 3, the OXA-23-Arg259, by a dual interaction, made polar contacts with the nitro-benzoyl carbonyl and boronic groups of compound (R)-6e. These bonds, stable over the majority of MD simulation frames, were mediated by water molecule bridges. Moreover, the OXA-23-Lys216 was hydrogen bound with the boronic group of (R)-6e, while the OXA-23-Trp113 and OXA-23-Phe110 interacted with the phenyl rings of (R)-6e by π–π (or Pi-Pi) stackings. Finally, the N-t-butyl group of (R)-6e was stabilized in a hydrophobic pocket sized by the Met221 and Trp219 residues of the β-lactamase.

The comparison between the predicted binding mode of 6e with the one found in the OXA-23/meropenem X-ray complex (see the Materials and Methods) suggested that the benzyl ring of the boronic compound should be substituted by a group with a lower size. In fact, during the MD simulations, a progressive opening of the catalytic crevice of the enzyme, for a repulsion between the benzyl ring and the side chain of OXA-23-Phe110, was observed (Figure 4). Moreover, a group substituting the p-nitro-phenyl ring, capable of interacting more efficiently with OXA-23-Arg259, could also ameliorate the affinity of the resulting compound.

3. Conclusions

In conclusion, structurally diverse, highly functionalized β-amino boronic acids have been efficiently synthesized via different multicomponent reactions. By means of the Ugi reaction, sixteen peptidomimetic β-amino boronic acids, functionalized with a variety of lipophilic and polar appendages, were prepared. The four centers-three components version of the Ugi reaction afforded three different β-lactam-based β-amino boronic acids, joining in a new scaffold two pharmacophoric moieties that are privileged in antibacterial drug discovery. Finally, the potential of the van Leusen reaction for the synthesis of imidazole-bearing methylboronic acids was demonstrated. The products were obtained in generally good yields, with simple workup procedures and straightforward isolation. They were fully characterized and a selection of them was subjected to biological evaluation, by determination of their in vitro antimicrobial susceptibility. By a molecular modelling investigation on the most active compound 6e, a plausible binding mode in the binding site of β-lactamase OXA-23 was proposed and compared with the one found in the OXA-23/meropenem X-ray complex, leading to suggestions for further insights aimed at improving the biological activity.

4. Materials and Methods

4.1. General Methods

All commercial materials and solvents (> 95% purity grade) were purchased from Merck KGaA (Darmstadt, Germany) and used without further purification. All reactions were carried out under a nitrogen atmosphere, unless otherwise noted. All reactions were monitored by thin layer chromatography (TLC) on precoated silica gel 60 F254; spots were visualized with UV light, or by treatment with a 1% aqueous KMnO₄ solution, or with a hydroalcoholic curcumin solution (100 mg of curcumin in a 100 mL solution of ethanol with 2 N HCl (99:1 v/v)). Products were purified by flash chromatography (FC) on silica gel 60 (230–400 mesh). Yields refer to isolated compounds estimated to be >95% pure as determined by ¹H-NMR. NMR spectra were recorded on 300 or 400 MHz Bruker spectrometers, using tetramethylsilane (TMS) as the internal standard. Supplementary materials for ¹³C-NMR, the APT pulse sequence was adopted. Chemical shifts are reported in parts per million relative to the residual solvent. Multiplicities in ¹H-NMR are reported as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br s = broad singlet. High-resolution MS spectra (HR-MS) were recorded with a Thermo Fisher LCQ Fleet ion trap mass spectrometer, equipped with an ESI source. The purity of compound 6e was confirmed to be >95% by means of elemental analysis on a CHN PerkinElmer 2400 instrument.

4.2. General Procedure for the Ugi-4CR (GP-A)

A test tube for the carousel was equipped with a magnetic stir bar and it was charged with compound 1 (120 mg, 0.6 mmol) and dry acetonitrile (6 mL). The primary amine 2a–d (0.7 mmol), the carboxylic acid 4a–c (0.7 mmol), and ammonium chloride (53 mg, 1.0 mmol) were added, and the reaction mixture was allowed to stir at 30 °C for 15 min. Then, the isocyanide 3a–e (1.0 mmol) was added in portions over a period of three h, after that the reaction mixture was kept at 60 °C for 21 h. The reaction was monitored by TLC (DCM/MeOH, 95:5, curcumin-based colorimetric method). The reaction mixture was concentrated in vacuo; then ethyl acetate (5 mL) was added. The solution was washed with saturated NaHCO₃ aq (×3), dried over Na₂SO₄, and concentrated under reduced pressure, to give a residue that was purified by a silica gravimetric chromatography column (eluent: DCM/MeOH = 99/1 to 96/4).

4.3. General Procedure for MIDA Deprotection (GP-B)

Under a nitrogen atmosphere, a round-bottom flask was equipped with a magnetic stir bar and charged with β-amino boronate 5a–p or 8a–c (0.5 mmol) and dry MeOH (7 mL). Solid Na₂CO₃ (9.0 mmol) was added. The resulting mixture was stirred for 4 h, and then the suspension was filtered and rinsed with a small amount of MeOH. The filtrate was concentrated in vacuo and the residue was partitioned between saturated aqueous NaHCO₃ and ethyl acetate. The layers were separated, and the aqueous layer was additionally extracted with ethyl acetate. Combined ethyl acetate layers were dried over Na₂SO₄ and concentrated in vacuo.

4.4. General Procedure for the Ugi-4C-3CR (GP-C)

A test tube for carousel was equipped with a magnetic stir bar and it was charged with compound 1 (100 mg, 0.5 mmol) and dry acetonitrile (5 mL). The β-amino acids 7a–c (0.6 mmol) and ammonium chloride (53 mg, 1.0 mmol) were added and the reaction was allowed to stir at 30 °C for 15 min. Then, tert-butyl isocyanide 3a (1.0 mmol) was added in portions over a period of three h; after that the reaction mixture was kept at 60 °C for 21 h. The reaction was monitored by TLC (DCM/MeOH, 95:5, curcumin-based colorimetric method). The reaction mixture was concentrated in vacuo and it was purified in a silica gravimetric chromatography column (eluent: DCM/MeOH = 99/1 to 95/5).

4.5. Procedure for the VL-3CR Reaction (GP-D)

A test tube for carousel was equipped with a magnetic stir bar, and it was charged with compound 1 (100 mg, 0.5 mmol) and dry DMF (5 mL). Cyclohexylmethanamine (0.08 mL, 0.6 mmol), was added and the resulting mixture was kept under stirring for 2 h at 50 °C under an atmosphere of nitrogen. Then, potassium carbonate (104 mg, 0.75 mmol) and TosMIC (148 mg, 0.75 mmol) were added in portions and the reaction was left overnight at 50 °C under stirring. The resulting mixture was concentrated in vacuo, and then it was partitioned between ethyl acetate/water. The aqueous phase was extracted with ethyl acetate (×3) and the organic phase was washed with brine (×5), dried over Na₂SO₄, and concentrated in vacuo, to give a residue that was purified by silica gravimetric chromatography column (eluent: DCM/MeOH = 99/1 to 90/10). The purified product was obtained with 32% yield as a brown solid.

N-(4-bromophenyl)-N-(1-(tert-butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)cinnamamide (5a): Prepared according to GP-A using α-borylaldehyde 1, 4-bromoaniline 2a, trans cinnamic acid 4a, and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 74%); ¹H-NMR (400 MHz, CD₃OD) δ 7.68–7.52 (m, 3H), 7.35–7.25 (m, 7H), 6.22 (d, J = 15.6 Hz, 1H), 5.38–5.21 (m, 1H), 4.17 (d, J = 17.1 Hz, 1H), 4.13 (d, J = 16.8 Hz, 1H), 4.00 (d, J = 17.1 Hz, 1H), 3.96 (d, J = 16.8 Hz, 1H), 2.99 (s, 3H), 1.36 (s, 9H), 1.18 (d, J = 14.4 Hz, 1H), 0.96 (dd, J = 14.4, 4.4 Hz, 1H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 171.3, 170.0, 169.4, 167.6, 143.2, 138.9, 135.4, 132.9, 132.8, 132.7 (2C), 132.4, 130.4, 129.3 (2C), 128.2 (2C), 123.0, 119.2, 62.6, 62.5, 58.1, 51.6, 46.0, 28.2 (3C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₃₁BBrN₃NaO₆ 606.1387; found 606.1399.

N-(1-(tert-Butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(4-methoxybenzyl)cinnamamide (5b): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, trans cinnamic acid 4a and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 51%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6) δ 7.54 (m, 3H), 7.37 (d, J = 7.2 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 7.00 (d, J = 16.0 Hz, 1H), 6.87 (d, J = 8.1 Hz, 2H), 6.75 (s, 1H), 6.48 (d, J = 16.0 Hz, 1H), 4.88–4.81 (m, 2H), 4.54 (d, J = 16.2 Hz, 1H), 4.14 (d, J = 16.9 Hz, 1H), 4.10 (d, J = 16.8 Hz, 1H), 3.96 (d, J = 16.9 Hz, 1H), 3.94 (d, J = 16.8 Hz, 1H), 3.74 (s, 3H), 2.95 (s, 3H), 1.34–1.27 (m, 1H), 1.17 (s, 9H), 0.95–0.85 (m, 1H); ¹³C-NMR (101 MHz, 25 °C, DMSO-d6, 6:4 rotameric mixture) δ 170.4–166.4 (4C), 158.5, 144.6, 141.7 and 141.5 (1C), 135.9 and 135.5 (1C), 131.8, 129.3 and 129.2 (2C), 128.6, 128.4, 128.2 and 128.1 (1C), 120.0, 114.2 (2C), 62.3–62.0 (2C), 55.5, 55.4, 50.6 and 50.5 (1C), 46.8, 46.3 and 46.1 (1C), 29.4, 28.6 and 28.5 (1C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₃₆BN₃NaO₇ 572.2544; found 572.2524.

N-(1-(tert-Butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(5,5-dimethylhexyl)cinnamamide (5c): Prepared according to GP-A using α-borylaldehyde 1, 5,5-dimethylhexan-1-amine 2c, trans cinnamic acid 4a and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 29%); ¹H-NMR (400 MHz, CD₃OD) δ 7.75 (d, J = 15.4 Hz, 1H), 7.71–7.61 (m, 5H), 7.06 (d, J = 15.4 Hz, 1H), 5.02 (m, J = 7.6 Hz, 0.7H), 4.50 (m, 0.3H), 4.17 (d, J = 17.1 Hz, 1H), 4.13 (d, J = 16.8 Hz, 1H), 4.02 (d, J = 17.1 Hz, 1H), 4.00 (d, J = 16.8 Hz, 1H), 3.67–3.53 (m, 2H), 3.08 (s, 2.1H), 3.01 (s, 0.9H), 1.73–1.58 (m, 4H), 1.45 (dd, J = 14.5, 8.8 Hz, 1H), 1.43–1.15 (m, 11H), 1.08 (dd, J = 14.5, 6.5 Hz, 1H), 0.90 (s, 9H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 171.5, 169.4, 168.8, 168.2, 142.7, 135.2, 130.2 and 129.6 (1C), 128.6 (2C), 128.0–127.6 (2C), 118.3, 61.8 (2C), 57.3, 55.9, 50.7, 45.3, 43.6, 31.4 (2C), 30.5, 28.4 (3C), 27.5 and 27.3 (3C), 21.9 (1C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₄₄BN₃NaO₆ 564.3221; found 564.3244.

N-(4-Bromophenyl)-N-(1-(tert-butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-4-nitrobenzamide (5d): Prepared according to GP-A using α-borylaldehyde 1, 4-bromoaniline 2a, 4-nitrobenzoic acid 4b and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 57%); ¹H-NMR (400 MHz, CD₃OD) δ 8.08 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2H), 5.25 (dd, J = 7.0 Hz, 1H), 4.22 (d, J = 17.1 Hz, 1H), 4.18 (d, J = 16.8 Hz, 1H), 4.03 (d, J = 17.1 Hz, 1H), 4.01 (d, J = 16.8 Hz, 1H), 3.02 (s, 3H), 1.38 (s, 9H), 1.26 (dd, J = 7.0, 4.2 Hz, 2H), (NH missed); ¹³C-NMR (75 MHz, CD₃OD, 2:1 rotameric mixture) δ 174.0–169.6 (4C), 149.7 and 149.6 (1C), 144.4 and 144.1 (1C), 140.9 and 140.2 (1C), 133.7–133.5 (4C), 130.6 (2C), 124.5 and 124.4 (2C), 123.5 and 123.3 (1C), 63.5, 63.4, 61.2 and 60.7 (1C), 58.7, 46.9, 29.2 (3C), (B-C_α missed); ¹¹B-NMR (128 MHz, CD₃CN) δ 12.26; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₅H₂₈BBrN₄NaO₈ 625.1081; found 625.1092.

N-Benzyl-N-(1-(tert-butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-4-nitrobenzamide (5e): Prepared according to GP-A using α-borylaldehyde 1, benzylamine 2d, 4-nitrobenzoic acid 4b and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 51%); ¹H-NMR (400 MHz, CD₃OD) δ 8.24 (d, J = 7.5 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.35–7.05 (m, 5H), 4.80 (d, J = 15.8 Hz, 1H), 4.71 (m, 1H), 4.48 (d, J = 15.8 Hz, 1H), 4.24 (d, J = 16.8 Hz, 1H), 4.22 (d, J = 16.4 Hz, 1H), 4.14–3.98 (m, 2H), 3.04 (s, 3H), 1.58 (d, J = 8.0 Hz, 1H), 1.35 (d, J = 8.0 Hz, 1H), 1.29 (s, 9H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 171.4–168.8 (4C), 148.2, 142.6, 137.0, 128.5 (2C), 128.2, 127.7 (2C), 127.4, 127.2, 123.3 (2C), 61.7, 61.5, 58.2, 57.3, 52.0, 45.1, 42.5, 27.4 (3C); ¹¹B NMR (128 MHz, CD₃CN) δ 12.33; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₆H₃₁BN₄NaO₈ 561.2133; found 561.2109.

N-(1-(tert-Butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(5,5-dimethylhexyl)-4-nitrobenzamide (5f): Prepared according to GP-A using α-borylaldehyde 1, 5,5-dimethylhexan-1-amine 2c, 4-nitrobenzoic acid 4b and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 40%); ¹H-NMR (400 MHz, CD₃OD) δ 8.36 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.5 Hz, 2H), 4.74 (dd, J = 9.6, 5.9 Hz, 1H), 4.21 (d, J = 17.1 Hz, 1H), 4.19 (d, J = 16.9 Hz, 1H), 4.05 (d, J = 17.1 Hz, 1H), 4.03 (d, J = 16.9 Hz, 1H), 3.42–3.21 (m, 2H), 3.07 (s, 3H), 1.53 (dd, J = 14.0, 9.6 Hz, 1H), 1.48–1.21 (m, 12H), 0.92 (m, 4H), 0.78 (s, 9H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 171.8, 171.3, 169.4, 168.9, 148.4, 142.8, 127.8 (2C), 123.5 (2C), 61.7, 61.6, 58.1, 57.1, 50.8, 45.2 (2C), 43.1, 30.0, 28.2 (3C), 27.5 (3C), 21.5 (2C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₄₁BN₄NaO₈ 583.2915; found 583.2931.

N-(4-Bromophenyl)-N-(1-(tert-butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)dec-9-enamide (5g): Prepared according to GP-A using α-borylaldehyde 1, 4-bromoaniline 2a, 9-decenoic acid 4c and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 50%); ¹H-NMR (400 MHz, CD₃OD) δ 7.62 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.3 Hz, 2H), 5.88–5.73 (m, J = 13.0 Hz, 1H), 5.20 (m, 1H), 4.95 (dd, J =13.0 Hz, 2H), 4.14–4.12 (m, 1H), 4.08 (d, J = 16.7 Hz, 1H), 3.97 (d, J = 17.6 Hz, 1H), 3.92 (d, J = 16.7 Hz, 1H), 2.95 (s, 3H), 2.34–2.26 (m, 1H), 2.09–1.97 (m, 4H), 1.65–1.48 (m, 3H), 1.36 (s, 9H), 1.27–1.13 (m, 6H), 1.04 (dd, J = 14.4, 10.5 Hz, 1H), 0.84 (dd, J = 14.4, 4.8 Hz, 1H), (NH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 176.1, 172.7, 171.0, 170.3, 140.4, 140.0, 133.9, 133.7, 133.5, 133.2, 123.9, 114.9, 63.6, 63.4, 58.79, 58.6, 46.9, 36.1 (2C), 35.1, 30.4 (3C), 29.2 (3C), 26.6 (2C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₈H₄₁BBrN₃NaO₆ 628.2169; found 628.2154.

N-(1-(tert-Butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(4-methoxybenzyl)dec-9-enamide (5h): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, 9-decenoic acid 4c and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 61%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6) δ 7.18 (d, J = 8.1 Hz, 2H), 6.85 (d, J = 8.1 Hz, 2H), 6.69 (s, 1H), 5.90–5.72 (m, 1H), 4.97 (dd, J = 14.4 Hz, 2H), 4.74–4.55 (m, 2H), 4.39 (d, J = 16.4 Hz, 1H), 4.16–4.03 (m, 2H), 4.00–3.88 (m, 2H), 3.75 (s, 3H), 2.93 (s, 3H), 2.40–2.17 (m, 2H), 2.09–1.95 (m, 2H), 1.54 (m, 2H), 1.44–1.23 (m, 8H), 1.18 (s, 9H), 0.93–0.81 (m, 2H); ¹³C-NMR (101 MHz, 25 °C, DMSO-d6, 1:1 rotameric mixture) δ 174.6–169.4 (4C), 159.2 and 158.8 (1C), 139.9, 133.0 and 132.0 (1C), 129.4, 128.6, 115.7, 114.8, 114.2, 63.0 and 62.6 (2C), 58.4 (0.5C), 56.2, 55.8 (0.5C), 51.3 and 51.1 (1C), 47.8, 46.9 and 46.8 (1C), 34.2 and 33.5 (2C), 30.1 and 29.5 (4C), 29.2 (3C), 25.9 and 25.7 (2C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₃₀H₄₆BN₃NaO₇ 594.3327; found 594.3320.

N-(1-(tert-Butylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(5,5-dimethylhexyl)dec-9-enamide (5i): Prepared according to GP-A using α-borylaldehyde 1, 5,5-dimethylhexan-1-amine 2c, 9-decenoic acid 4c and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 38%); ¹H-NMR (400 MHz, DMSO-d6, 6:4 rotameric mixture) δ 7.48 (s, 0.4H), 7.04 (s, 0.6H), 5.92–5.68 (m, 1H), 4.96 (dd, J =14.2 Hz, 2H), 4.74 (t, J = 7.1 Hz, 0.6H), 4.28 (dd, J = 8.6, 5.0 Hz, 0.4H), 4.21–4.06 (m, 2H), 4.04–3.87 (m, 2H), 3.29–3.19 (m, 1.2H), 3.17–3.04 (m, 0.8H), 2.96 (s, 1.8H), 2.90 (s, 1.2H), 2.33–2.22 (m, 2H), 2.03–1.93 (m, 2H), 1.59–1.39 (m, 4H), 1.37–1.32 (m, 2.4H), 1.30–1.07 (m, 19.8H), 0.85 (s, 5.5H), 0.84 (s, 3.5H), 0.80 (d, J = 7.1 Hz, 0.3H), 0.67 (dd, J = 14.4, 5.0 Hz, 0.5H); ¹³C-NMR (101 MHz, DMSO-d6, 6:4 rotameric mixture) δ 174.0, 172.8 and 172.1 (1C), 170.9, 170.0 and 169.6 (1C), 139.9, 115.7, 62.8–62.5(2C), 57.9 and 55.7 (1C), 51.4 and 51.0 (2C), 46.8 and 46.7 (1C), 44.7 and 44.5 (1C), 34.3- 31.2 (6C), 30.3 (3C), 29.4 (3C), 26.1 and 25.9 (2C), 23.2 and 22.9 (2C); ¹¹B-NMR (128 MHz, CD₃CN) δ 12.56; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₃₀H₅₄BN₃NaO₆ 586.4003; found 586.4024.

N-(1-(Cyclohexylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(4-methoxybenzyl)cinnamamide (5j): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, trans cinnamic acid 4a and cyclohexyl isocyanide 3b. Obtained as a white solid (yield = 49%); ¹H-NMR (400 MHz, CD₃OD) δ 7.67 (d, J = 6.9 Hz, 1H), 7.61 (d, J = 15.5 Hz, 1H), 7.46 (d, J = 3.6 Hz, 1H), 7.40 (t, J = 3.6, 6.9 Hz, 2H), 7.35 (d, J = 3.6 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 15.5 Hz, 1H), 6.90 (d, J = 8.6 Hz, 2H), 5.13–5.05 (m, 1H), 4.95 (d, J = 17.1 Hz, 1H), 4.74 (d, J = 17.1 Hz, 1H), 4.17 (d, J = 17.1 Hz, 1H), 4.13 (d, J = 17.3 Hz, 1H), 3.99 (d, J = 17.1 Hz, 1H), 3.97 (d, J = 17.3 Hz, 1H), 3.77 (s, 3H), 3.52–3.44 (m, 1H), 3.03 (s, 2H), 2.98 (s, 1H), 1.89–1.80 (m 1H), 1.79–1.73 (m, 1H), 1.73–1.64 (m, 2H), 1.62–1.52 (m, 2H), 1.47–1.20 (m, 5H), 1.16–1.09 (m, 1H), (NH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 172.4–169.7 (4C), 160.8, 144.6, 136.7, 131.91, 131.3, 130.2 (2C), 129.5 (2C), 129.3 (2C), 120.2, 115.5 (2C), 63.5 (2C), 57.5, 56.1, 52.3, 47.0, 43.3, 33.7 (2C), 31.0, 26.9, 26.2 (2C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₃₁H₃₈BN₃NaO₇ 598.2701; found 598.2689.

N-(1-(Cyclohexylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(4-methoxybenzyl)-4-nitrobenzamide (5k): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, 4-nitrobenzoic acid 4b, and cyclohexyl isocyanide 3b. Obtained as a white solid (yield = 53%); ¹H-NMR (400 MHz, CD₃OD, 6:4 rotameric mixture) δ 8.27 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 2H), 7.04 (d, J = 7.9 Hz, 2H), 6.82 (d, J = 7.9 Hz, 2H), 4.74–4.62 (m, 1.4H), 4.57 (s, 0.2H), 4.42 (d, J = 15.5 Hz, 0.6H), 4.25 (d, J = 17.1 Hz, 1H), 4.23 (d, J = 16.7 Hz, 1H), 4.13 (d, J = 17.1 Hz, 1H), 4.11 (d, J = 16.7 Hz, 1H), 4.06 (m, 0.8H), 3.77 (s, 3H), 3.64–3.47 (m, 1H), 3.04 (s, 3H), 1.87–1.55 (m, 6H), 1.46–1.22 (m, 6H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 170.9–168.0 (4C), 159.5, 148.3, 148.2, 129.2 (2C), 128.6, 127.8 (2C), 123.4 (2C), 113.9 (2C), 61.7, 61.6, 57.4, 54.4, 51.7, 45.2, 41.7, 32.1 (2C), 29.3, 25.2 (2C), 24.5; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₃₅BN₄NaO₉ 617.2395; found 617.2382.

N-(4-Methoxybenzyl)-N-(3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxo-1-(pentylamino)propan-2-yl)cinnamamide (5l): Prepared according to GP-A using α-borylaldehyde 2, 4-methoxybenzylamine 3b, trans cinnamic acid 4a, and 1-pentyl isocyanide 5e. Obtained as a white solid (yield = 40%); ¹H-NMR (400 MHz, CD₃OD, rotameric mixture) δ 7.80–7.51 (m, 2H), 7.49–7.31 (m, 4H), 7.29–7.21 (m, 2H), 7.13–6.79 (m, 3H), 5.14 (m, 0.5H), 5.00–4.91 (m, 0.6H), 4.71 (m, 1.9H), 4.18 (d, J = 16.8 Hz, 1H), 4.14 (d, J = 16.0 Hz, 1H), 4.00 (d, J = 16.8 Hz, 1H), 3.95–3.90 (m, 1H), 3.81–3.73 (m, 3H), 3.24–3.15 (m, 0.5H), 3.14–2.78 (m, 4.5H), 1.76–1.05 (m, 7H), 1.07–0.73 (m, 4H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD, rotameric mixture) δ 172.4–168.6 (4C), 159.7, 144.9 and 143.5 (1C), 135.9 and 135.7 (1C), 131.2 and 130.8 (1C) 130.7–129.7 (2C), 129.3 and 129.2 (2C), 128.5 and 128.4 (2C), 128.2, 119.2, and 117.8 (1C), 114.6 and 114.4 (2C), 62.5, 62.5, 56.3, 55.0, 51.3, 46.0, 40.7 and 39.9 (1C), 29.5, 29.1 (2C), 22.7, 13.6; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₃₀H₃₈BN₃NaO₇ 586.2701; found 586.2719.

N-(4-Methoxybenzyl)-N-(3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxo-1-(pentylamino)propan-2-yl)-4-nitrobenzamide (5m): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, 4-nitrobenzoic acid 4b, and 1-pentyl isocyanide 3c. Obtained as a white solid (yield = 53%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6, 9:1 rotameric mixture, only the major rotamer reported here) δ 8.17 (d, J = 7.8 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.24 (s, 1H), 7.11 (s, 2H), 6.78 (d, J = 7.8 Hz, 2H), 4.66–4.51 (m, 2H), 4.46 (d, J = 15.6 Hz, 1H), 4.15 (d, J = 15.3 Hz, 1H), 4.12 (d, J = 15.6 Hz, 1H), 3.97 (d, J = 15.3 Hz, 1H), 3.93 (d, J = 15.6 Hz, 1H), 3.73 (s, 3H), 3.01 (d, J = 6.0 Hz, 2H), 2.91 (s, 3H), 1.47–1.21 (m, 8H), 0.89 (t, J = 6.0 Hz, 3H); ¹³C-NMR (101 MHz, 25 °C, DMSO-d6, 7:3 rotameric mixture) δ 170.9–168.8 (4C), 158.6 and 158.3 (1C), 148.0 and 147.7 (1C), 144.3 and 143.7 (1C), 131.2 and 129.9 (1C), 129.1 (2C), 128.8, 128.0, 124.0, 123.7, 113.9, 113.7, 62.1 (2C), 55.5, 55.4, 49.9, 46.2, 39.3, 29.4, 29.1 (2C), 22.3, 14.4; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₈H₃₅BN₄NaO₉ 605.2395; found 605.2380.

N-(1-(Benzylamino)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-N-(4-methoxybenzyl)-4-nitrobenzamide (5n): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, 4-nitrobenzoic acid 4b, and benzyl isocyanide 3d. Obtained as a white solid (yield = 43%); ¹H-NMR (400 MHz, CD₃OD) δ 8.24 (d, J = 7.7 Hz, 2H), 7.75 (d, J = 7.7 Hz, 2H), 7.40–7.17 (m, 5H), 6.99 (d, J = 7.2 Hz, 2H), 6.69 (d, J = 7.2 Hz, 2H), 4.77–4.69 (m, 1H), 4.66 (d, J = 15.3 Hz, 1H), 4.41 (d, J = 15.3 Hz, 1H), 4.33 (s, 1.6H), 4.25 (d, J = 17.1 Hz, 1H), 4.23 (d, J = 16.2 Hz, 1H), 4.15 (d, J = 17.1 Hz, 0.2H), 4.13 (d, J = 17.1 Hz, 1H), 4.07 (d, J = 16.2 Hz, 1H), 3.97 (d, J = 17.1 Hz, 0.2H), 3.73 (s, 3H), 3.03 (s, 3H), 1.42–1.24 (m, 2H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 171.9, 171.0, 169.4, 168.9, 168.0, 159.3, 148.1, 142.8, 138.3, 129.3 (2C), 128.2 (2C), 127.7 (2C), 127.3, 126.8 (2C), 123.3 (2C), 113.7 (2C), 61.7, 61.5, 57.1, 54.3, 51.9, 45.2, 43.0, 29.3; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₃₀H₃₁BN₄NaO₉ 625.2082; found 625.2069.

Methyl (2-(N-(4-methoxybenzyl)cinnamamido)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)propanoyl)glycinate (5o): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, trans cinnamic acid 4a, and methyl isocyanoacetate 3e. Obtained as a brown solid (yield = 38%); ¹H-NMR (300 MHz, CD₃OD, 8:2 rotameric mixture) δ 7.73–7.51 (m, 2H), 7.48–7.18 (m, 6H), 7.02–6.78 (m, 3H), 5.23 (m, 0.8H), 4.92 (m, 1.2H), 4.72 (d, J = 17.2 Hz, 0.8H), 4.62 (d, J = 14.8 Hz, 0.2H), 4.14 (d, J = 17.1 Hz, 1H), 4.11 (d, J = 16.7 Hz, 1H), 3.98 (d, J = 17.1 Hz, 1H), 3.96 (d, J = 16.7 Hz, 1H), 3.83 (d, J = 12.0 Hz, 2H), 3.74 (s, 3H), 3.69 (s, 3H), 2.98 (s, 2.4H), 2.88 (s, 0.6H), 1.70–1.53 (m, 0.2H), 1.44 (dd, J = 14.4, 8.3 Hz, 0.8H), 1.14 (dd, J = 14.4, 6.6 Hz, 0.8H), 1.05–0.93 (m, 0.2H), (NH missed); ¹³C-NMR (75 MHz, CD₃OD, rotameric mixture) δ 177.4–171.8 (5C), 163.3 and 162.9 (1C), 148.8 and 146.8 (1C), 139.0 and 138.5 (1C), 134.1, 133.9, 133.5, 132.6 and 132.4 (2C), 131.9 and 131.8 (2C), 131.5, 122.4 and 120.7 (1C), 117.9 and 117.7 (2C), 65.9, 65.8, 63.0, 60.9, 58.3, 55.2, 45.6, 44.7, 33.3; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₈H₃₂BN₃NaO₉ 588.2129; found 588.2114.

Methyl (2-(N-(4-Methoxybenzyl)-4-nitrobenzamido)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)propanoyl)glycinate (5p): Prepared according to GP-A using α-borylaldehyde 1, 4-methoxybenzylamine 2b, 4-nitrobenzoic acid 4b, and methyl isocyanoacetate 3d. Obtained as a brown solid (yield = 42%); ¹H-NMR (300 MHz, 115 °C, DMSO-d6) δ 8.19 (d, J = 8.2 Hz, 2H), 7.78 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.25–7.05 (m, 2H), 6.79 (d, J = 7.9 Hz, 2H), 4.82–4.56 (m, 2H), 4.46 (d, J = 15.8 Hz, 1H), 4.17 (d, J = 17.0 Hz, 1H), 4.14 (d, J = 17.1 Hz, 1H), 3.97 (d, J = 17.0 Hz, 1H), 3.95 (d, J = 17.1 Hz, 1H), 3.83 (dd, J = 10.1, 5.5 Hz, 2H), 3.75 (s, 3H), 3.70 (s, 3H), 2.91 (s, 3H), 1.46–1.12 (m, 2H); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, rotameric mixture) δ 171.3–168.4 (5C), 158.2 and 157.9 (1C), 147.7 and 147.3 (1C), 143.8 and 143.1 (1C), 130.8, 128.9 (2C), 128.4, 127.6, 123.5 and 123.4 (2C), 113.5 and 113.3 (2C), 61.6 (2C), 59.3, 55.1, 51.8, 49.5, 45.8, 40.9, (B-C_α missed); ¹¹B-NMR (128 MHz, CD₃CN) δ 12.17; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₆H₂₉BN₄NaO₁₁ 607.1824; found 607.1828.

(2-(N-(4-Bromophenyl)cinnamamido)-3-(tert-butylamino)-3-oxopropyl)boronic acid (6a): Prepared according to GP-B starting from 5a. Obtained as a white solid (yield = 97%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6) δ 7.63 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 15.5 Hz, 1H), 7.40–7.28 (m, 7H), 6.92 (s, 1H), 6.26 (d, J = 15.5 Hz, 1H), 5.20 (dd, J = 9.4, 6.1 Hz, 1H), 1.29 (s, 9H), 0.98 (dd, J = 15.2, 9.4 Hz, 1H), 0.78 (dd, J = 15.2, 6.1 Hz, 1H), (BOH missed); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, 6:4 rotameric mixture) δ 172.7 and 171.5 (1C), 164.9, 141.6 and 141.1 (1C), 138.6, 134.5, 132.6, 132.0 (2C), 129.9 and 129.8 (1C), 129.0 (3C), 127.7 (2C), 121.5, 119.5 and 119.0 (1C), 58.8 and 56.9 (1C), 51.2 and 50.1 (1C), 28.5 and 28.3 (3C), 19.3; ¹¹B-NMR (128 MHz, CD₃CN) δ 32.01; HR-MS (ESI) m/z: [C₂₂H₂₄BrN₂O₂B(OMe)₂ + Na]⁺ calcd for C₂₄H₃₀BBrN₂NaO₄ 523.1380; found 523.1391.

(3-(tert-butylamino)-2-(N-(4-methoxybenzyl)cinnamamido)-3-oxopropyl)boronic acid (6b): Prepared according to GP-B starting from 5b. Obtained as a white solid (yield = 78%); ¹H-NMR (400 MHz, CD₃OD) δ 7.74 (d, J = 15.4 Hz, 1H), 7.60–7.55 (m, 2H), 7.41–7.37 (m, 3H), 7.27 (d, J = 8.5 Hz, 2H), 7.11 (d, J = 15.4 Hz, 1H), 6.94 (d, J = 8.5 Hz, 2H), 5.02 (d, J = 16.7 Hz, 1H), 4.70–4.62 (m, 2H), 3.79 (s, 3H), 1.37–1.27 (m, 9H), 1.18–1.14 (m, 1H), 0.88–0.80 (m, 1H), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 176.0, 169.8, 161.2, 146.1, 136.4, 131.8, 130.5, 130.3 (2C), 129.9 (2C), 129.6 (2C), 118.7, 115.6 (2C), 62.9, 56.1, 53.8, 52.9, 31.1, 28.9 (3C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.55; HR-MS (ESI) m/z: [C₂₄H₂₉N₂O₃B(OMe)₂ + Na]⁺ calcd for C₂₆H₃₅BN₂NaO₅ 489.2537; found 489.2549.

(3-(tert-Butylamino)-2-(N-(5,5-dimethylhexyl)cinnamamido)-3-oxopropyl)boronic acid (6c): Prepared according to GP-B starting from 5c. Obtained as a white solid (yield = 61%); ¹H-NMR (400 MHz, CD₃OD) δ 7.75 (d, J = 15.4 Hz, 1H), 7.71–7.59 (m, 2H), 7.44 (m, 3H), 7.11 (d, J = 15.4 Hz, 1H), 4.50 (m, 1H), 3.99–3.72 (m, 1H), 3.53–3.35 (m, 1H), 1.78–1.48 (m, 2H), 1.48–1.11 (m, 15H), 0.90 (s, 9H), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 171.2, 170.2, 146.2, 134.1, 131.9, 130.4 (2C), 129.7 (2C), 118.2, 63.4, 54.3, 52.3, 45.3, 32.1, 31.0, 30.0 (3C), 29.0 (3C), 23.2, (B-C_α missed); ¹¹B-NMR (128 MHz, CD₃CN) δ 30.99; HR-MS (ESI) m/z: [C₂₄H₃₇N₂O₂B(OMe)₂ + Na]⁺ calcd for C₂₆H₄₃BN₂NaO₄ 481.3214; found 481.3234.

(2-(N-(4-Bromophenyl)-4-nitrobenzamido)-3-(tert-butylamino)-3-oxopropyl)boronic acid (6d): Prepared according to GP-B starting from 5d. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6, 1:1 rotameric mixture) δ 8.12–7.96 (m, 2H), 7.53 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.45–7.38 (m, 2H), 7.28–7.18 (m, 2H), 6.91 (s, 1H), 5.29–5.13 (m, 1H), 1.32 (s, 9H), 1.10 (dd, J = 15.5, 9.0 Hz, 0.5H), 1.04 (dd, J = 14.1, 8.1 Hz, 0.5H), 0.97 (dd, J = 8.1, 5.1 Hz, 0.5H), 0.91 (dd, J = 15.5, 6.8 Hz, 0.5H), (BOH missed); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, 2:1 rotameric mixture) δ 170.9, 167.7, 147.4 and 147.2 (1C), 143.4, 138.8, 132.5 (2C), 131.9 and 131.8 (1C), 131.5 (2C), 128.9, 123.1 (2C), 120.8 and 120.7 (1C), 57.7, 50.3, 28.5 and 28.2 (3C), (B-C_α missed); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.06; HRMS (ESI) m/z: [C₂₀H₂₁BrN₃O₄B(OMe)₂ + Na]⁺ calcd for C₂₂H₂₇BBrN₃NaO₆ 542.1074; found 542.1061.

(2-(N-Benzyl-4-nitrobenzamido)-3-(tert-butylamino)-3-oxopropyl)boronic acid (6e): Prepared according to GP-B starting from 5e. Obtained as a white solid (yield = 89%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6, 7:3 rotameric mixture) δ 8.27–8.16 (m, 2H), 7.82 (s, 0.3H), 7.67 (d, J = 8.6 Hz, 0.6H), 7.61 (d, J = 8.6 Hz, 1.4H), 7.30–7.16 (m, 5H), 6.59 (s, 0.7H), 4.77 (dd, J = 16.3, 9.2 Hz, 1H), 4.72–4.55 (m, 1H), 4.51 (dd, J = 16.3, 9.2 Hz, 1H), 1.30 (dd, J = 15.5, 6.8 Hz, 1H), 1.24 (s, 9H), 1.13 (dd, J = 15.5, 6.8 Hz, 1H), (BOH missed); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, rotameric mixture) δ 171.2–170.2 (2C), 147.6, 143.6, 139.0, 128.0 (2C), 127.7 (2C), 126.8 (2C), 126.3, 123.7 (2C), 59.9 and 59.4 (1C), 50.3 and 50.0 (1C), 46.5, 28.3 (3C), 18.5; HR-MS (ESI) m/z: [C₂₁H₂₄N₃O₄B(OMe)₂ + Na]⁺ calcd for C₂₃H₃₀BN₃NaO₆ 478.2125; found 478.2104. Anal. calcd for C₂₁H₂₆BN₃O₆: C, 59.03; H, 6.13; B, 2.53; N, 9.83; O, 22.47; found: C, 59.33; H, 6.25; N, 9.56.

(3-(tert-Butylamino)-2-(N-(5,5-dimethylhexyl)-4-nitrobenzamido)-3-oxopropyl)boronic acid (6f): Prepared according to GP-B starting from 5f. Obtained as a white solid (yield = 95%); ¹H-NMR (400 MHz, CD₃OD) δ 8.38 (d, J = 8.1 Hz, 2H), 7.69 (d, J = 8.1 Hz, 2H), 4.75–4.63 (m, 1H), 3.43–3.24 (m, 2H), 1.69–1.52 (m, 2H), 1.50–1.23 (m, 12H), 1.14–0.99 (m, 3H), 0.81 (s, 9H), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 172.0, 171.8, 150.3, 143.7, 129.3 (2C), 125.3 (2C), 61.3, 51.2, 45.1 (2C), 31.9, 31.2, 29.9 (3C), 29.0 (3C), 23.3 (2C); ¹¹B NMR (128 MHz, CD₃CN) δ 31.22; HR-MS (ESI) m/z: [C₂₂H₃₄N₃O₄B(OMe)₂ + Na]⁺ calcd for C₂₄H₄₀BN₃NaO₆ 500.2908; found 500.2924.

(2-(N-(4-Bromophenyl)dec-9-enamido)-3-(tert-butylamino)-3-oxopropyl)boronic acid (6g): Prepared according to GP-B starting from 5g. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6) δ 7.59 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 7.12 (s, 1H), 5.87–5.73 (m, 1H), 5.09 (dd, J = 9.9, 5.8 Hz, 1H), 4.96 (dd, J = 21.4, 13.7 Hz, 2H), 2.08–1.89 (m, 4H), 1.51–1.42 (m, 2H), 1.37–1.14 (m, 17H), 0.86 (dd, J = 14.9, 9.9 Hz, 1H), 0.66 (dd, J = 14.9, 5.8 Hz, 1H), (BOH missed); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, rotameric mixture) δ 172.7 and 172.2 (1C), 172.0 and 171.5 (1C), 139.1, 138.8, 132.4 (2C), 131.9 (2C), 121.3 and 121.1 (1C), 114.6, 56.2, 50.0, 34.2, 33.1, 28.5, 28.4 (3C), 28.3, 28.2, 28.1, 24.8, 17.2; ¹¹B-NMR (128 MHz, CD₃CN) δ 31.21; HR-MS (ESI) m/z: [C₂₃H₃₄BrN₂O₂B(OMe)₂ + Na]⁺ calcd for C₂₅H₄₀BBrN₂NaO₄ 545.2162; found 545.2150.

(3-(tert-Butylamino)-2-(N-(4-methoxybenzyl)dec-9-enamido)-3-oxopropyl)boronic acid (6h): Prepared according to GP-B starting from 5h. Obtained as a white solid (yield = 74%); ¹H-NMR (400 MHz, CD₃OD, 8:2 rotameric mixture) δ 7.22 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.2 Hz, 2H), 5.91–5.73 (m, 1H), 5.06–4.88 (m, 2H), 4.76 (d, J = 16.7 Hz, 1H), 4.57–4.45 (m, 2H), 3.80 (s, 3H), 2.61–2.41 (m, 2H), 2.12–1.96 (m, 2H), 1.79–1.52 (m, 2H), 1.52–1.18 (m, 17H), 1.10 (dd, J = 14.0 Hz, 1H), 0.79 (dd, J = 14.0 Hz, 1H), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 179.5, 177.9, 163.4, 142.6, 134.8, 132.2, and 131.91 (2C), 117.9 (2C), 117.3, 64.4, 58.3, 55.7, 55.0, 37.3(2C), 33.3–32.5 (4C), 31.1 (3C), 28.9 (2C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.54; HR-MS (ESI) m/z: [C₂₅H₃₉N₂O₃B(OMe)₂ + Na]⁺ calcd for C₂₇H₄₅BN₂NaO₅ 511.3319; found 511.3334.

(3-(tert-Butylamino)-2-(N-(5,5-dimethylhexyl)dec-9-enamido)-3-oxopropyl)boronic acid (6i): Prepared according to GP-B starting from 5i. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6) δ 7.11 (s, 1H), 5.90–5.73 (m, 1H), 4.97 (dd, J = 23.7, 13.7 Hz, 2H), 4.80–4.53 (m, 1H), 3.34–3.17 (m, 2H), 2.32 (t, J = 6.6 Hz, 2H), 2.04 (d, J = 6.3 Hz, 2H), 1.63–1.43 (m, 6H), 1.43–1.11 (m, 20H), 0.88 (s, 9H), 0.79 (m, 1H), (BOH missed); ¹³C-NMR (75 MHz, 25 °C, CD₃OD) δ 173.1, 172.3, 138.9, 114.7, 57.6, and 55.1 (1C), 44.6, 43.3 (2C), 33.2 (2C), 32.6 (2C), 30.7 (1C) 30.0 (1C), 29.2 (3C), 28.7 (2C), 28.3 (3C), 25.1 and 24.8 (2C), 22.1 and 21.7 (1C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.19; HR-MS (ESI) m/z: [C₂₅H₄₇N₂O₂B(OMe)₂ + Na]⁺ calcd for C₂₇H₅₃BN₂NaO₄ 503.3996; found 503.4006.

(3-(Cyclohexylamino)-2-(N-(4-methoxybenzyl)cinnamamido)-3-oxopropyl)boronic acid (6j): Prepared according to GP-B starting from 5j. Obtained as a white solid (yield = 83%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6, 6:4 rotameric mixture) δ 7.65 (d, J = 7.1 Hz, 1H), 7.58–7.46 (m, 3H), 7.43 (d, J = 7.1 Hz, 1H), 7.39–7.29 (m, 2H), 7.24–7.13 (m, 2H), 6.96–6.79 (m, 2H), 5.18–5.12 (m, 0.4H), 4.84 (d, J = 17.6 Hz, 0.6H), 4.77–4.69 (m, 0.6H), 4.62 (d, J = 15.2 Hz, 0.4H), 4.56 (d, J = 17.6 Hz, 0.6H), 4.49 (d, J = 15.2 Hz, 0.4H), 3.70 (s, 3H), 3.48–3.37 (m, 1H), 1.64–1.44 (m, 4H), 1.32 (dd, J = 15.2 Hz, 1H), 1.25–1.00 (m, 6H), 0.85 (dd, J = 15.2, 5.9 Hz, 0.5H), 0.77 (dd, J = 15.2, 5.9 Hz, 0.5H), (NH, BOH missed); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, 6:4 rotameric mixture) δ 170.5 and 169.9 (1C), 166.5 and 166.2 (1C), 158.1 and 158.0 (1C), 141.4, 135.0, and 135.0 (1C), 131.3 and 131.2 (2C), 129.6, 128.8 (3C), 127.8 (2C), 127.6, 119.6, 113.8, 113.5, 55.5, 55.0, 47.8, and 47.6 (1C), 47.1, 32.1 (2C), 29.0, 25.2, 24.3 (2C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.40; HR-MS (ESI) m/z: [C₂₆H₃₂N₂O₄B(OMe) + Na]⁺ calcd for C₂₇H₃₅BN₂NaO₅ 501.2537; found 501.2549.

(3-(Cyclohexylamino)-2-(N-(4-methoxybenzyl)-4-nitrobenzamido)-3-oxopropyl)boronic acid (6k): Prepared according to GP-B starting from 5k. Obtained as a white solid (yield = 66%); ¹H-NMR (400 MHz, CD₃OD, 6:4 rotameric mixture) δ 8.45–8.25 (m, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 7.4 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H), 6.94–6.76 (m, 2H), 5.27 (d, J = 16.2 Hz, 0.4H), 4.79–4.64 (m, 0.6H), 4.64–4.50 (m, 1.2H), 4.46 (d, J = 16.2 Hz, 0.4H), 4.41–4.29 (m, 0.4H), 3.81 (s, 1.2H), 3.78 (s, 1.8H), 3.71–3.48 (m, 1H), 1.93–1.70 (m, 4H), 1.70–1.54 (m, 2H), 1.47–1.05 (m, 5H), 1.05–0.74 (m, 1H), (NH, BOH missed); ¹³C NMR (75 MHz, CD₃OD, 6:4 rotameric mixture) δ 174.0 and 173.6 (1C), 169.6 and 169.5 (1C), 161.3 and 160.7 (1C), 150.2, 144.4 and 143.6 (1C), 132.2, 130.5, 130.1, 129.5, 129.1, 125.2 (2C), 115.5, 115.2, 61.4, and 60.8 (1C), 56.0, 54.2, 51.6, and 50.6 (1C), 33.8 (2C), 31.0, 26.8, 26.3 (2C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.58; HR-MS (ESI) m/z: [C₂₄H₂₈N₃O₅B(OMe)₂ + Na]⁺ calcd for C₂₆H₃₄BN₃NaO₇ 534.2388; found 534.2373.

(2-(N-(4-Methoxybenzyl)cinnamamido)-3-oxo-3-(pentylamino)propyl)boronic acid (6l): Prepared according to GP-B starting from 5l. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, DMSO-d6, rotameric mixture) δ 7.62–7.26 (m, 7H), 7.18 (d, J = 7.7 Hz, 2H), 6.88 (m, 3H), 5.29–5.07 (m, 0.6H), 4.82 (d, J = 17.2 Hz, 0.6H), 4.79–4.73 (m, 0.4H), 4.70 (d, J = 17.2 Hz, 0.2H), 4.55 (t, J = 8.1 Hz, 1.2H), 3.72 (s, 3H), 3.16–2.85 (m, 2H), 1.53–0.99 (m, 6H), 0.99–0.61 (m, 5H), (BOH missed); ¹³C-NMR (75 MHz, DMSO-d6, rotameric mixture) δ 171.3 and 170.9 (1C), 166.4 and 165.1 (1C), 158.5 and 158.1 (1C), 141.8 and 141.1 (1C), 135.3 and 135.0 (1C), 131.1 and 130.3 (1C), 129.6, 128.8 (2C), 128.4, 128.0 and 127.8 (2C), 127.5, 119.6 and 118.7 (1C), 113.9 and 113.4 (2C), 55.6, 55.0, 47.3, 28.6 (3C), 21.9, 15.7, 13.9; ¹¹B NMR (128 MHz, CD₃CN) δ 31.20; HR-MS (ESI) m/z: [C₂₅H₃₁N₂O₃B(OMe)₂ + Na]⁺ calcd for C₂₇H₃₇BN₂NaO₅ 503.2693; found 503.2704.

(2-(N-(4-Methoxybenzyl)-4-nitrobenzamido)-3-oxo-3-(pentylamino)propyl)boronic acid (6m): Prepared according to GP-B starting from 5m. Obtained as a white solid (yield = 64%); ¹H-NMR (400 MHz, CD₃OD, 6:4 rotameric mixture) δ 8.41–8.26 (m, 2H), 7.74 (d, J = 7.2 Hz, 2H), 7.31 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H), 6.94–6.82 (m, 2H), 5.26 (d, J = 15.7 Hz, 0.4H), 4.73–4.65 (m, 0.5H), 4.55 (dd, J = 15.7 Hz, 1.2H), 4.45–4.41 (m, 0.5H), 4.37 (d, J = 15.8 Hz, 0.4H), 3.81 (s, 1.3H), 3.78 (s, 1.7H), 3.28–3.21 (m, 1.1H), 3.19–3.08 (m, 0.9H), 1.61–1.42 (m, 2H), 1.42–1.22 (m, 6H), 0.96–0.90 (m, 3H), (NH, BOH missed); ¹³C-NMR (101 MHz, CD₃OD, rotameric mixture) δ 176.1, 173.4, 162.5, 149.2, 143.2, 136.0, 129.2 (2C), 128.3 (2C), 124.0 (2C), 114.4 (2C), 61.6 and 61.0 (1C), 55.1, 54.5, 40.3, 29.9, 29.4, 29.2, 22.6. (B-C_α missed); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.59; HR-MS (ESI) m/z: [C₂₃H₂₈N₃O₅B(OMe)₂ + Na]⁺ calcd for C₂₅H₃₄BN₃NaO₇ 522.2388; found 522.2401.

(3-(Benzylamino)-2-(N-(4-methoxybenzyl)-4-nitrobenzamido)-3-oxopropyl)boronic acid (6n): Prepared according to GP-B starting from 5n. Obtained as a white solid (yield = 97%); ¹H-NMR (400 MHz, CD₃OD) δ 8.31–8.17 (m, 2H), 7.64 (d, J = 5.9 Hz, 2H), 7.41–7.23 (m, 6H), 7.06 (d, J = 7.3 Hz, 1H), 6.91 (d, J = 7.7 Hz, 1H), 6.81 (d, J = 7.3 Hz, 1H), 4.62–4.21 (m, 5H), 3.81 (s, 3H), 1.44–1.19 (m, 2H), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 169.8, 169.4, 167.0, 158.9, 150.1, 142.8, 138.2, 130.3, 130.2, 129.9 (2C), 129.1 (2C), 128.7 (2C), 125.1 (3C), 115.4, 115.3, 61.6, 56.1, 53.7, 44.6, 31.0; ¹¹B-NMR (128 MHz, CD₃CN) δ 30.99; HR-MS (ESI) m/z: [C₂₅H₂₄N₃O₅B(OMe)₂ + Na]⁺ calcd for C₂₇H₃₀BN₃NaO₇ 542.2075; found 542.2058.

(3-((2-Methoxy-2-oxoethyl)amino)-2-(N-(4-methoxybenzyl)cinnamamido)-3-oxopropyl)boronic acid (6o): Prepared according to GP-B starting from 5o. Obtained as a brown solid (yield = 99%); ¹H-NMR (400 MHz, CD₃OD) δ 7.79 (d, J = 15.3 Hz, 1H), 7.59–7.53 (m, 2H), 7.43–7.34 (m, 3H), 7.26 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 15.3 Hz, 1H), 6.94 (d, J = 8.2 Hz, 2H), 5.03–4.93 (m, 2H), 4.74 (d, J = 17.2 Hz, 1H), 3.95 (d, J = 9.4 Hz, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 1.03–0.86 (m, 2H), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD) δ 175.2, 171.9, 170.5, 161.1, 146.5, 136.3, 131.8, 130.3 (2C), 129.6 (2C), 129.5 (2C), 128.6, 118.6, 115.6 (2C), 60.6, 56.0, 52.9, 51.9, 42.4, 31.0; ¹¹B-NMR (128 MHz, CD₃CN) δ 31.50; HR-MS (ESI) m/z: [C₂₃H₂₅N₂O₅B(OMe)₂ + Na]⁺ calcd for C₂₅H₃₁BN₂NaO₇ 505.2122; found 505.2135.

(3-((2-Methoxy-2-oxoethyl)amino)-2-(N-(4-methoxybenzyl)-4-nitrobenzamido)-3-oxopropyl)boronic acid (6p): Prepared according to GP-B starting from 5p. Obtained as a brown solid (yield = 91%); ¹H-NMR (400 MHz, CD₃OD, rotameric mixture) δ 8.44–8.22 (m, 2H), 7.91–7.64 (m, 2H), 7.46–6.75 (m, 4H), 5.38 (d, J = 14.8 Hz, 0.3H), 5.22–4.97 (m, 0.4H), 4.74–4.64 (m, 0.3H), 4.64–4.44 (m, 1.3H), 4.41–4.31 (m, 0.7H), 4.02–3.89 (m, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 1.71–1.48 (m, 1H), 1.48–1.25 (m, 1H), (NH, BOH missed); ¹³C-NMR (101 MHz, CD₃OD, rotameric mixture) δ 172.6, 171.8, 170.9, 159.7, 149.1, 143.2, and 142.9 (1C), 131.2, 129.3, and 129.1 (2C), 128.4 (2C), 124.0 (2C), 114.2 (2C), 60.6, 55.0, 52.0, 42.6, 41.2, 30.0; ¹¹B NMR (128 MHz, CD₃CN) δ 31.57; HR-MS (ESI) m/z: [C₂₁H₂₂N₃O₇B(OMe)₂ + Na]⁺ calcd for C₂₃H₂₈BN₃NaO₉ 524.1816; found 524.1827.

N-(tert-Butyl)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-2-(2-oxoazetidin-1-yl)propenamide (8a): Prepared according to GP-C using α-borylaldehyde 1, β-alanine 7a and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 72%); ¹H-NMR (400 MHz, CD₃OD) δ 4.35 (dd, J = 9.1, 6.2 Hz, 1H)), 4.18 (d, J = 17.1 Hz, 1H), 4.17 (d, J = 16.9 Hz, 1H), 4.02 (d, J = 17.1 Hz, 1H), 3.99 (d, J = 16.9 Hz, 1H), 3.47 (dd, J = 8.4, 4.9 Hz, 1H), 3.41 (dd, J = 8.4, 4.9 Hz, 1H), 3.01 (s, 3H), 2.94–2.86 (m, 2H), 1.36 (s, 9H), 1.22 (dd, J = 14.4, 9.1 Hz, 1H), 1.13 (dd, J = 14.4, 6.2 Hz, 1H), (NH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 171.8 and 171.7 (1C), 169.9, 169.5 (2C), 62.3 and 62.2 (2C), 54.3, 53.8, 51.5, 38.4, 35.3, 30.0, 28.1 (3C); ¹¹B-NMR (128 MHz, CD₃CN) δ 12.15; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₁₅H₂₄BN₃NaO₆ 376.1656; found 376.1647.

N-(tert-Butyl)-3-(4-methyl-2,6-dioxotetrahydro-2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-2-((S)-2-oxo-4-phenylazetidin-1-yl)propenamide (8b): Prepared according to GP-C using α-borylaldehyde 1, S-β-phenylalanine 7b and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 36%); ¹H-NMR (400 MHz, CD₃OD, 3:2 diasteroisomeric mixture, presence of rotamers) δ 7.58–7.15 (m, 5H), 4.86 (dd, J = 5.3, 2.3 Hz, 0.1H), 4.78 (dd, J = 5.3, 2.3 Hz, 0.3H), 4.70 (ddd, J = 10.8, 5.3, 2.3 Hz, 0.6H), 4.32–4.28 (m, 0.2H), 4.18–4.11 (m, 2.6H), 4.02–3.94 (m, 2H), 3.87–3.83 (m, 0.2H), 3.44–3.35 (m, 1H), 3.00 (s, 1.8H), 2.99 (s, 1.2H), 2.83–2.75 (m, 1H), 1.55–1.06 (m, 11H), (NH missed); ¹³C-NMR (75 MHz, CD₃OD, 3:2 diasteroisomeric mixture, presence of rotamers) δ 173.2–170.5 (4C), 140.6 and 140.4 (1C), 130.2 (2C), 129.9 and 129.7 (1C), 128.3 (2C), 63.5, 63.4, and 63.3 (1C), 57.3–57.0 (1C), 47.3 and 46.2 (1C), 46.9 and 46.7 (1C), 32.1, 31.0, 29.1, and 29.0 (3C), 24.0; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₂₁H₂₈BN₃NaO₆ 452.1969; found 452.1985.

Methyl (2S)-1-(1-(tert-butylamino)-3-(4-methyl-2,6-dioxotetrahydro -2H-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborol-8-yl)-1-oxopropan-2-yl)-4-oxoazetidine-2-carboxylate (8c): Prepared according to GP-C using α-borylaldehyde1, (3S)-3-ammonio-4-methoxy-4- oxobutanoate 7c and tert-butyl isocyanide 3a. Obtained as a white solid (yield = 52%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6, 1:1 diasteroisomeric mixture) δ 7.71 (s, 0.5H), 7.56 (s, 0.5H), 4.38–4.33 (m, 0.5H), 4.21–4.13 (m, 3H), 4.06–3.94 (m, 2.5H), 3.72 (s, 1.5H), 3.61 (s, 1.5H), 3.12 (dd, J = 14.2, 5.8 Hz, 1H), 2.91 (s, 1.5H), 2.89 (s, 1.5H), 2.78 (dd, J = 14.2, 2.0 Hz, 1H), 1.26 (s, 4.5H), 1.22 (s, 4.5H), 1.15 (d, J = 8.1 Hz, 0.5H), 1.10–0.99 (m, 1H), 0.93 (dd, J = 14.6, 8.1 Hz, 0.5H); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, 1:1 diasteroisomeric mixture) δ 172.1 and 171.3 (1C), 169.0, 168.8, 168.6, 165.8, and 165.0 (1C), 61.8 (2C), 55.2 and 52.3 (1C), 52.1 and 51.7 (1C), 51.0 and 49.0 (1C), 50.2 and 50.1 (1C), 45.7, 41.4, and 40.8 (1C), 29.0, 28.3 (3C); HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₁₇H₂₆BN₃NaO₈ 434.1711; found 434.1723.

(3-(tert-Butylamino)-3-oxo-2-(2-oxoazetidin-1-yl)propyl)boronic acid (9a): Prepared according to GP-B starting from 8a. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, CD₃OD) δ 4.35 (t, J = 7.8 Hz, 1H, CH), 3.41–3.30 (m, 2H, CH₂-N), 2.92–2.86 (m, 2H, CH₂-CO), 1.35 (s, 9H, tert-Bu), 1.21 (dd, J = 15.7, 7.8 Hz, 2H, CH₂-B), (NH, BOH missed); ¹³C-NMR (101 MHz, CD₃OD) δ 172.1 (CO), 169.3 (CO), 54.3 (CH-N), 51.7 ((CH₃)₃C-N), 38.1 (CH₂-N), 35.3 (CH₂-CO), 30.0 (CH₂-B), 28.0 (3C, (CH₃)₃C); ¹¹B-NMR (128 MHz, CD₃OD) δ 28.84; HR-MS (ESI) m/z: [M + Na]⁺ calcd for C₁₀H₁₉BN₂NaO₄ 265.1336; found 265.1357.

(3-(tert-Butylamino)-3-oxo-2-((S)-2-oxo-4-phenylazetidin-1-yl)propyl)boronic acid (9b): Prepared according to GP-B starting from 8b. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, 115 °C, DMSO-d6, 6:4 diastereoisomeric mixture) δ 7.48–7.26 (m, 5H, Ph), 6.87 (s, 0.4H, NH), 6.71 (s, 0.6H, NH), 4.83–4.75 (m, 0.4H, CH-Ph), 4.72–4.62 (m, 0.6H, CH-Ph), 4.12–3.99 (m, 1H, CH), 3.28 (dd, J = 14.5, 5.4 Hz, 1H, CH-CO), 2.74–2.63 (m, 1H, CH-CO), 1.44–1.11 (m, 10H, tert-Bu, CH-B), 0.98 (d, J = 7.4 Hz, 1H, CH-B), (BOH missed); ¹³C-NMR (75 MHz, 25 °C, DMSO-d6, 6:4 diastereoisomeric mixture) δ 170.6 and 169.9 (1C, CO), 167.5 and 167.1 (1C, CO), 140.3 and 139.6 (1C, C(Ph)), 128.6 (2C, CH(Ph)), 127.9 (CH(Ph)), 126.6 (2C, CH(Ph)), 54.8–52.9 (1C, CH-N), 50.1 ((CH₃)₃C-N), 45.7 (CH₂-CO), 31.6–29.5 (1C, CH-Ph), 29.0 (CH₂-B), 28.4 and 28.2 (3C, (CH₃)₃C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.09; HR-MS (ESI) m/z: [C₁₆H₂₁N₂O₂B(OMe)₂ + Na]⁺ calcd for C₁₈H₂₇BN₂NaO₄ 369.1962; found 369.1970.

(3-(tert-Butylamino)-2-((S)-2-(methoxycarbonyl)-4-oxoazetidin-1-yl)-3-oxopropyl)boronic acid (9c): Prepared according to GP-B starting from 8c. Obtained as a white solid (yield = 99%); ¹H-NMR (400 MHz, CD₃OD, 1:2 diastereoisomeric mixture) δ 4.42-4.21 (m, 2H, CH-N, CH-CO₂), 3.83 and 3.80 (s, 3H, OCH₃), 3.23 (dd, J = 14.4, 5.6 Hz, 1H, CH-CO), 2.94 (q, J = 14.4, 11.8 Hz, 1H, CH-CO), 1.47–1.21 (m, 11H, tert-Bu, CH₂-B), (NH, BOH missed); ¹³C-NMR (75 MHz, CD₃OD, 1:2 diastereoisomeric mixture) δ 173.6 (CO₂), 172.8 (CO), 169.5 (CO), 57.4 and 55.9 (1C, CH-N), 53.4 and 53.2 (1C, CH-CO₂), 52.8 ((CH₃)₃C-N), 51.8 (CH₃O), 42.3 (CH₂-CO), 31.0 (CH₂-B), 29.0 (3C, (CH₃)₃C); ¹¹B-NMR (128 MHz, CD₃CN) δ 31.24; HR-MS (ESI) m/z: [C₁₂H₁₉N₂O₄B(OMe)₂+Na]⁺ calcd for C₁₄H₂₅BN₂NaO₆ 351.1703; found 351.1724.

8-((1-(Cyclohexylmethyl)-1H-imidazol-5-yl) methyl)-4-methyldihydro-4λ⁴,8λ⁴-[1,3,2]oxazaborolo[2,3-b][1,3,2]oxazaborole-2,6(3H,5H)-dione (10): Prepared according to GP-D. Obtained as a brown solid (yield = 32%); ¹H-NMR (300 MHz, (CD₃)₂CO) δ 7.65 (s, 1H, CH(Im)), 6.81 (s, 1H, CH(Im)), 4.27 (d, J = 16.9 Hz, 2H, CH₂-N(MIDA)), 4.05 (d, J = 16.9 Hz, 2H, CH₂-N(MIDA)), 3.88 (d, J = 7.4 Hz, 2H, CH₂-N(Im)), 3.18 (s, 3H, N-CH₃), 1.88–1.52 (m, 9H, CH and 4 CH₂(Cy)), 1.26–1.15 (m, 2H, CH₂(Cy)), 1.12–0.93 (m, 2H, CH₂-B); ¹³C-NMR (101 MHz, CD₃OD) δ 169.4 (2C, CO₂), 130.7 (CH(Im)), 129.3 (C(Im)), 118.1 (CH(Im)), 61.5 (2C, CH₂-N (MIDA)), 52.4 and 52.1 (1C, CH₂-N), 42.2 (CH₃-N (MIDA)), 38.5 (CH(Cy)), 30.5 (2C, CH₂(Cy)), 30.0 (CH₂(Cy)), 26.5 (CH₂(Cy)), 25.9 (2C, CH₂(Cy), CH₂-B); HR-MS (ESI) m/z: [M + H]⁺ calcd for C₁₆H₂₅BN₃O₄ 334.1938; found 334.1927.

Single crystal X-ray diffraction analysis of compounds 5a and 6d: Single crystals of 5a and 6d were obtained by slow evaporation from the mother liquor. All X-ray data collections were performed at room temperature with a Bruker AXS Smart 3-circle diffractometer equipped with an APEX-II CCD detector. Graphite–monochromated Mo Kα radiation (λ = 0.71073 Å) at a nominal power of 50 kV × 30 mA of the sealed X-ray tube was employed. Highly-redundant ω-scans (Δω = 0.25 deg) at variable φ angles were performed, resulting in 100% complete spheres of data up to 2θ = 46.5 deg (5a) and 2θ = 52.7 deg (6d). Diffraction patterns were corrected by absorption and beam anisotropy using SADABS [31], and then phased by direct methods with Shelx [32]. Both compounds crystallized in centrosymmetric space groups (5a: P2₁/c; 6d: P$\overline{1}$) as 1:1 racemates. The interested reader can find full details of the diffraction analysis in the Supporting Information.

4.6. In Vitro Antibacterial Susceptibility Assays

The potential of each molecule described herein to increase the activity of a β-lactam antibiotic was investigated using recombinant isogenic E. coli strains producing different types of β-lactamases, including the class A ESBL CTX-M-15 and the KPC-2 carbapenemase, the Enterobacter cloacae AmpC enzyme, the plasmid-encoded class C CMY-2, and several OXA-typ enzymes (OXA-10 and the carbapenemases OXA-23, OXA-40, and OXA-48). The β-lactamase genes were cloned in the pLB-II vector as previously described [33], and subsequently used to transform E. coli DH5α. In addition, clinical isolates present in our collection of antibiotic-resistant clinical isolates were also tested. Minimal inhibitory concentrations (MICs) of ampicillin, imipenem, and cefepime were determined in triplicate by the broth microdilution method in Mueller-Hinton broth, according to the Clinical Laboratory Standards Institute guidelines [34], in the absence and presence of 16 or 32 μg/mL of the tested compound. Plates were incubated aerobically at 35 ± 1 °C for 18–24 h before reading.

4.7. Molecular Modeling Studies, Model System, and MD Setup

The OXA-23/Mer complex computational model was built taking into account the crystal structures acquired at different pH values (Protein Data Bank entries 4JF4 and 4JF6) [35,36], as reported on our previous paper in which the meropenem hydrolysis mechanism was predicted by QM/MM simulations [37]. Here, the previously optimized OXA-23 model was fully solvated without the presence of meropenem into the catalytic site, in order to be ready for the covalent docking of the compound under investigation. The TIP3P [38] model was employed to describe the solvent molecules’ effect, and the OPLS3e force fields [39] were applied to simulate the enzyme atoms. The neutrality of the system was ensured by adding two sodium ions; in fact, after the calculation of the enzyme charge, the “protein preparation tool” of Maestro inserted the sodium ions on the protein surface where the negative charge was the highest. Then, MD simulations were accomplished by performing the following steps: (1) 100 ps of Brownian dynamics under isocore conditions (NVT) at a temperature of 10 K, restraining the enzyme heavy atoms; (2) 12 ps long MD simulations NVT, at the same temperature, while restraining the solute heavy atoms; (3) 12 ps long MD simulations in an isothermal–isobaric ensemble (NPT) at a temperature of 10 K, with restraints on the solute heavy atoms; (4) 24 ps of MD simulations in NPT conditions with no restraints; (5) 250 ns long MD simulations in NPT conditions. Finally, by visual inspection with VMD, [40] we ensured that the thermalization and the MD simulations did not cause any structural distortion.

4.8. Covalent Docking

The structure of compound 6e was drawn by the “build” tool of Maestro software and prepared for docking by the LigPrep module of Maestro (2019-4 release). This tool generates both enantiomers of the ligand, checks and corrects any geometrical distortions, assigns the OPLS3e force field to the atoms, and performs the energy minimization of the ligand model. Covalent docking on Ser79 residue of OXA-23 was performed by “CovDock” module of Glide [41]. The default parameters of docking were adopted for this calculation in which the boronic acid’s electrophile attack on the reactive residue Ser79 was simulated. The attained docking results highlighted that the R enantiomer of compound 6e acquired the highest score. Thus, the complex composed by the docking solution with the highest score in the catalytic site of OXA-23 was submitted to geometrical optimization and MD simulations, following the protocol previously adopted for the OXA-23 model. Finally, the MD trajectory was carefully analyzed by the “simulation interactions diagram” module of Maestro software, in order to evaluate the stability of the ligand binding pose in the catalytic site of the β-lactamase.