Direct Diazo-Transfer Reaction on β-lactam: Synthesis and Preliminary Biological Activities of 6-Triazolylpenicillanic Acids

Abstract

In this study we report the first example of a direct diazo-transfer reaction on readily available 6-aminopenicillanates to give 6-azidopenicillanates in high yield. Subsequent Cu(I)-catalyzed Huisgen cycloaddition between these 6-azidopenicillanates and assorted terminal alkynes facilely furnished 6-triazolylpenicillanic acids was. Preliminary biological screening indicates that these triazolylpenicillanic acids possess low to moderate antibacterial activities.

Introduction

β-lactam antibiotics such as penicillin and cephalosporins are the most commonly prescribed antibacterial agents. Their antibacterial efficacy derives from ability to acylate a serine residue at the penicillin binding proteins (PBPs) active site.^{1, 2} Serine acylation inhibits the enzymatic activities of PBPs, thus interfering with the essential polymerization and cross-linking of the peptidoglycan components of the bacterial cell wall.¹ However, the emergence of multi-drug resistant bacteria strains such as Staphylococcus aureus is threatening the clinical effectiveness of β-lactam antibiotics.³ The common resistance mechanism in gram-negative bacteria is the cellular expression of β-lactamases (or penicillinase), which hydrolyze the β-lactam ring and thus inactivate the antibiotics.^4–7 Therapeutic agents targeting β-lactamase resistance include clavulanate, sulbactam, and tazobactam. These are generally administered in combination with β-lactamase susceptible β-lactams.^{8, 9} However, the emergence of multi-strains β-lactamase has reduced the efficacy of some of these β-lactamase inhibitors.^{10, 11}

There are also other intracellular factors that attenuate the antibacterial activities of β-lactams. For example, the N-5 amide moiety in penicillins is highly reactive and has been proposed to facilitate the hydrolysis of the β-lactam ring in acidic media (Figure 1).¹² Incorporation of electron-withdrawing groups and steric bulk at the N-acyl side chain has been shown to reduce the acid sensitivity of penicillins.^{13, 14}

Figure 1.

Self-induced acid hydrolysis of penicillin.

Another shortcoming to the effectiveness of β-lactamase antibiotics is the bacterial outer membrane, which is a formidable barrier in gram negative organisms. In addition, the bacterial membrane could acquire efflux pumps that facilitate active elimination of drug from the bacterial cytosol.^{15, 16} All these changes could prevent the intracellular accumulation of pharmacologically relevant concentrations of the β-lactam antibiotics. One clever strategy to facilitate β-lactams diffusion across membrane is to covalently link them to membrane penetrating groups such as sideophores and peptides.^{1–3, 17} However, the synthesis of such β-lactam conjugates often involve long synthetic schemes and complicated compound purifications protocols.2b–e

One of the most fruitful β-lactam SAR studies has been the modification of the N-acyl groups. These studies have led to the discovery of β-lactam analogs with superior antibacterial activity.^{16, 18–20} Much recent studies have shown that attachment of N-acyl groups incorporating the structural feature of peptidoglycan result in PBP specific β-lactams and cephalosporins.^{3, 17, 21} PBPs can also tolerate an assorted variety of non-amide groups at the C-6 and C-7 positions of penicillins and cephalosporins, respectively.²⁰ Some of these non-amide β-lactam analogs, in addition to possessing potent antibacterial activity, have differential affinity for PBP isoforms (Figure 3). For example, mecillinam (amdinocillin), a β-lactam analog with an unusual 6-formylimido moiety, derives its antibacterial activity from its specific interaction with PBP-2.²² Very few β-lactams with traditional amide bond isosteres have been reported to date. One such example is the sulfonamide derivatives. A subset of these compounds have been synthesized and tested. However these compounds are still prone to chemical degradation and hydrolysis by β-lactamases.²³

Figure 3.

Structure of amide isosteric β-lactams.

We proposed that 1,4-disubstituted-1,2,3-triazoles could serve as an isostere for the N-acyl group of penicillins and cephalosporins. Due to the similarities between some of its bond characteristics with an amide moeity, the triazole group is commonly used as an amide bond isostere.^{24, 25} Unlike amide bond, the triazole moiety is not susceptible to hydrolysis.²⁴ Replacement of the reactive β-lactam acyl bond with a triazole ring may lead to β-lactams that are more stable in acidic environment. In addition, the triazole ring could in principle facilitate facile conjugation of β-lactams to assorted cell permeable peptides and peptoids for promoted uptake of β-lactams into bacterial cell. Here, we report our efforts on the design, synthesis, and preliminary biological activities of 6-triazolylpenicillanic acid.

Results and Discussion

The key intermediate in the synthesis of the proposed 6-triazolylpenicillanic acids 1 is the 6-azidopenicillanates 2. It is anticipated that Cu(I)-catalyzed cycloaddition between 2 and appropriate alkynes (Sharpless Click Chemistry)²⁶ followed by deprotection of the ester protection group will furnish the desired triazolylpenicillanic acid 1 (scheme 1). The synthesis of protected 6-azidopenicillanates similar to 2a–c has been described in the literature.^27–30 These compounds are generally synthesized, in moderate yields, using multi-step reactions. For example, Barrett and Sakadarat described the synthesis of benzyl protected 6-azidopecillanate, the final intermediate in their total synthesis of 6-aminopenicillanic acid (6-APA), in a six-step reaction scheme with a total yield of about 15%.³⁰ For the synthesis of 6-triazolylpenicillanates, we desired a more general and mild synthetic strategy to 6-azidopenicillanates.

Scheme 1.

Synthetic approach to 6-triazolylpenicillanic acids. Abbreviation: PNB = para-nitrobenzyl, PMB = para-methoxylbenzyl, BzD = benzhydryl.

Synthesis of 6-Azidopenicillanic Acid by Diazo-Transfer Reaction

As a new general alternative, we envisioned that azidopenicillanic acid could be obtained from commercially available 6-aminopenicillanic acids through a diazo-transfer reaction. The diazo-transfer reaction is a mild, high yielding reaction that has been used to effect direct conversion of assorted amines to azides.³¹ In addition, a recent observation from our lab has extended the scope of this versatile reaction to include azide functionalization of amine-coated solid supports under heterogeneous reaction conditions.³² However, our initial efforts at initiating diazo-transfer reaction on unprotected 6-aminopenicillanic acid were unsuccessful. To thoroughly probe the feasibility of this reaction on β-lactam moiety, we decided to investigate various carboxyl protected 6-aminopenicillanates. The requisite carboxyl protected aminopenicillanates 3a–c were synthesized adapting literature protocols.^{21, 33–37} Using the PNB protected compound 3a, we initiated diazo-transfer reaction with freshly prepared triflyl azide under basic conditions in the classic CH₂Cl₂/MeOH/H₂O solvent mixture.31b–e These conditions, however, gave a complex mixture from which we only isolated a minute amount of the ring-opened methyl ester azide 4. This product was presumably obtained from Et₃N promoted methanolysis of compound 3a in addition to the desired diazo-transfer reaction. The reaction was repeated in anhydrous CH₂Cl₂ and Et₃N. Gratifyingly, we obtained the desired azide 5a in yields of 50–68% within 2 to 2.5 h of reaction, though other uncharacterized degradation products persisted. Because of the potential hazard of handling triflyl azide in halogenated solvent, we investigated the compatibility of this reaction with non-halogenated solvents. The reaction worked equally well when CH₂Cl₂ was replaced with toluene, yielding azide 5a in 70% yields. Similarly, azides 5b and 5c were obtained from amines 2b and 2c in moderate to good yields (scheme 2). The β orientation of the azide group in 5a–c was authenticated by ¹H NMR (J_5,6 coupling constant = 4 Hz),²⁹ thereby confirming that the reaction occurred with the characteristic retention of configuration.31b,e

Scheme 2.

Synthesis of protected 6-azidopenicillanic acid. Conditions: (a) TfN₃, Et₃N, CH₂Cl₂/MeOH/H₂O, rt, (b) TfN₃, Et₃N, DMAP, rt, 2–2.5 hrs, (c) TfN₃, Et₃N, toluene, rt, 1.5–2.5 hrs.

Cu(I)-catalyzed Cycloaddition Reaction between 6-Azidopenicillanates and Terminal Alkynes

To identify optimum conditions for Cu(I)-catalyzed cycloaddition reaction, we investigated the reaction of azides 5b and 5c with 3-phenyl-1-propyne 6 and 2-ethynyl-1,3-dimethoxybenzene 7. Our choice of terminal alkynes 6 and 7 is partly informed by the possibility that cycloaddition between these alkynes and azides 5 will respectively furnish triazolyl isosteres of penicillin G and methicillin, two historically useful penicillin derivatives. Cu(I)-catalyzed reaction of azide 5b or 5c with alkyne 6 resulted in triazoles 8a and 8b in excellent yield. Subsequent TFA deprotection of the PMB and the BzD group furnished the desired penicillanic acid 9a in excellent yield. Similarly, the reaction of azide 5c with alkyne 7 followed by deprotection of the BzD group gave penicillanic acid 9 in 72% overall yield (scheme 3). Because of the rapidity of deprotection of the BzD group and the product quality, we focused much of our attention on the reactivity of azide 5c.

Scheme 3.

Cu(I)-catalyzed cycloadditions reaction between 6-azidopenicillanates and representative terminal alkynes. Conditions: (a) CuI, Hunig’s base, alkynes 6 or 7, THF, rt, (b) TFA, anisole, CH₂Cl₂, −5°C. Abbreviation: 2,6-DMP = 2,6-dimethoxylphenyl.

We turned to investigate the reactivity of azide 5c with assorted terminal alkynes in order to probe the scope of this reaction. We selected a subset of terminal alkynes whose closely related carboxylic acid analogs have been shown to support the antimicrobial activities of β-lactams.^38–40 Alkynes such as 7, 10h, 10i, and 10k that we could not obtain from commercial sources were synthesized from the corresponding carboxylic acid, through the intermediacy of aldehyde, using the Bestmann-Ohira reagent.^41–43 Hydroxyl alkyne 10f and 10l were obtained by a direct Grignard reaction of ethynylmagnesium bromide with the appropriate aldehydes. Detailed protocols for the synthesis of these alkynes are reported in the experimental section.

Cu(I)-catalyzed reaction of alkynes 10a–l with azidopenicillanate 5b or 5c proceeded smoothly at ambient temperature, leading to uneventful formation of triazole 11a–l in good to excellent yields. Subsequent carbonyl group deprotection furnished the desired 6-triazolylpenicillanic acid 12a–l (scheme 4).

Scheme 4.

Scope of Cu(I)-catalyzed cycloaddition between 6-azidopenicillanate and terminal alkynes. Conditions: a) CuI, Hunig’s base, alkynes 10, THF, rt, b) TFA, anisole CH₂Cl₂, −5°C.

Biological Evaluation

The synthesized 6-triazolylpenicillanic acids were screened at the National Institute of Allergy and Infectious Disease (NIAID) through the In vitro and Animal Models for Emerging Infectious Diseases and Biodefense Screening Program. Compounds were screened following the procedures recommended by the Clinical Laboratory Standard Institute (CLSI). Compound 9a, 12e, 12g, and 12l exhibited some antibacterial activity against the Gram-positive bacteria strain S. pneumoniae with MIC value of 4 to 8 μg/mL. Similarly, compound 12l showed a moderate activity (MIC ≈ 8 μg/mL) against B. anthracis. Other compounds listed in Table 1 displayed comparatively poor antibacterial activity (MIC > 8μg/mL).

Table 1.

Minimal Inhibitory Concentration (MIC) of 6-triazolylepenicillanic acids in μg/mL: Each MIC values were obtained from an average of three independent experiments.

Compound	R2	S. aureus ATCC 29213 (μg/mL)	E. coli ATCC 25922 (μg/mL)	S. pneumoniae ATCC 49619 (μg/mL)	B. anthracis AMES (μg/mL)
9a		>8	>8	4	>8
9b		>8	>8	>8	>8
12a		>8	>8	>8	>8
12b		>8	>8	>8	>8
12c		>8	>8	>8	>8
12d		>8	>8	8	>8
12e		>8	>8	4	>8
12f		>8	>8	>8	>8
12g		>8	>8	≥8**	>8
12h		>8	>8	>8	>8
12i		>8	>8	>8	>8
12j		>8	>8	>8	>8
12k		>8	>8	>8	>8
12l*		>8	>8	≥8**	≥8**
		1	>2	N. D.***	≤0.125
		2	4	N. D.***	1

^*X = Na

^**Denotes that one of the 3 replicates had MIC value of 8 μg/mL.

^***No Data

Conclusion

We have reported a convenient route for the synthesis of 6-triazolyl-penicillanic acid. Preliminary biological evaluations of compounds described in this report demonstrated that they possess low to moderate antibacterial activity. The triazole ring lacks the H-bonding donating N-H group which could act as an additional recognition feature in the binding of some β-lactams to the PBPs. This may partly explain the relatively low antimicrobial activities of these 6-triazolyl-penicillanic acids. Currently, we are investigating the SAR of the lead compounds in order to identify 6-triazolylpenicillanic acid with improved antibacterial activity against drug resistant bacterial strains.

Experimental Section

6-aminopenicillanoic acid (6-APA) was purchased from Sigma Aldrich. Anhydrous solvents and other reagents were purchased and used without further purification. Analtech silica gel plates (60 F₂₅₄) were used for analytical TLC, and uv light was used to examine the spots. 200–400 Mesh silica gel was used in column chromatography. NMR spectra were recorded on a Varian-Gemini 400 magnetic resonance spectrometer. ¹H NMR spectra are recorded in parts per million (ppm) relative to the peak of CDCl₃, (7.24 ppm), acetone-d₆ (2.09 ppm), or DMSO-d₆ (2.49 ppm). ¹³C spectra were recorded relative to the central peak of the CDCl₃ triplet (77.0 ppm), acetone-d₆ (205.8 ppm), or the DMSO-d₆ septet (39.7 ppm), and were recorded with complete hetero-decoupling. High-resolution mass spectra were recorded at the Georgia Institute of Technology mass spectrometry facility in Atlanta. Triflyl azide was prepared as described before and used without storage.31h, 31i The Bestmann-Ohira reagent was prepared as described by Ghosh et al⁴² while diphenyldiazomethane was prepared by using the procedure described by Ko et al.³⁷ 6-Aminopenicillanates 3a–c were synthesized adapting literature procedures.^{21, 33–37}

Representative Procedure for the Alkyne Transformation Reaction. 2-Ethynyl-1,3-dimethoxybenzene (7)

2,6-dimethoxybenzaldehyde (0.45 g, 2.71 mmol) was first dissolved in anhydrous MeOH (25 mL) and stirred under argon at room temperature. Anhydrous K₂CO₃ (1.12 g, 8.12 mmol) and Bestmann-Ohira reagent (1.03 g, 5.42 mmol) were added to the reaction mixture and stirring continued for 24 h at room temperature. Solvent was evaporated off, and the remaining residue was dissolved in CH₂Cl₂ (25 mL) and washed with saturated NH₄Cl (3 × 20 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, 5:1 Hexane/EtOAc) to give 220 mg (50%) of 7 as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 3.55 (1H, s), 3.88 (6H, s), 6.53 (2H, d, J = 8.4 Hz), 7.24 (1H, t, 7.4 Hz); ¹³C-NMR (CDCl₃, 100MHz) δ 56.0, 76.2, 85.7, 100.0, 103.5, 130.3, 162.0.

4-(4-Ethynylphenyl)pyridine (10k)

Reaction of 4-(4-formylphenyl)pyridine (0.3 g, 1.64 mmol) and Bestmann-Ohira reagent (0.627 g, 3.27 mmol) within 24 h as described for the synthesis of compound 7 followed by flash chromatography (silica gel, 2:1 Hexane/EtOAc) gave 261 mg (89%) of 10k as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 3.16 (1H, s), 7.47 (2H, d, J = 6.4 Hz), 7.59 (4H, s), 8.66 (2H, d, J = 6.4Hz). HRMS (EI) calc for [C₁₃H₉N] 179.0735, found 179.0746.

2-Ethnylbiphenyl (10i)

The required Biphenyl-2-carbaldehyde was synthesized from the corresponding carboxylic acid through NaBH₄/BF₃:OEt₂ reduction to the primary alcohol.⁴³ Subsequent PDC oxidation of the primary alcohol furnished Biphenyl-2-carbaldehyde. Reaction of biphenyl-2-carbaldehyde (0.66 g, 3.63 mmol) and Bestmann-Ohira reagent (1.72 g, 7.27 mmol) within 24 h followed by flash chromatography (silica gel, 5:1 Hexane/EtOAc) yielded 523 mg (81%) of 10i as a colorless oil. ¹H-NMR (CDCl₃, 400MHz) δ 3.03 (1H, s), 7.28–7.32 (1H, m), 7.36–7.45 (5H, m), 7.57–7.63 (3H, m).

3-Ethynylbiphenyl (10h)

The required Biphenyl-3-carbaldehyde was synthesized from the corresponding carboxylic acid through NaBH₄/BF₃:OEt₂ reduction to the primary alcohol.⁴³ Subsequent PDC oxidation of the primary alcohol furnished Biphenyl-3-carbaldehyde. Reaction of biphenyl-3-carbaldehyde (0.87 g, 4.78 mmol) and Bestmann-Ohira reagent (1.72 g, 9.56 mmol) within 24 h followed by flash chromatography (silica gel, 5:1 Hexane/EtOAc) gave 850 mg (100%) of 10h as a reddish oil. ¹H-NMR (CDCl₃, 400MHz) δ 3.03 (1H, s), 7.33–7.47 (5H, m), 7.55–7.57 (3H, m), 7.72 (1H, s). HRMS(EI) calc for [C₁₄H₁₀] 178.0782, found 178.0797.

Representative Procedure for the Alkyne Transformation via Grignard Reaction. 1-phenylprop-2-yn-1-ol (10f)

To a solution of benzaldehyde (0.5 g, 4.71 mmol) in anhydrous THF (2 mL) was added ethynylmagnesium bromide (14.0 mL, 0.5M in THF) at room temperature under argon. The reaction mixture was stirred for 1 h and quenched with distilled water. The reaction mixture was partitioned between CH₂Cl₂ (10 mL) and water (15 mL), and the two layers separated. The organic layer was washed in succession with distilled water (2 × 10 mL) and saturated brine (2 × 10 mL), and dried over Na₂SO₄. Solvent was evaporated off to give 0.626g (100%) of 10f as a brownish oil. ¹H-NMR (CDCl₃, 400MHz) δ 2.66 (1H, d, J = 2.4 Hz), 5.45 (1H, d, J = 4.0 Hz), 7.30–7.40 (3H, m), 7.54 (2H, d, J = 7.2 Hz); HRMS (FAB, thioglycerol) calc for [C₉H₈O] 132.0575, found 132.0566.

1-(4-(pyrdin-4-yl)prop-2-yn-1-ol (10l)

To a solution of benzaldehyde (0.5 g, 2.73 mmol) in anhydrous THF (2 mL) was added ethynylmagnesium bromide (8.2 mL, 0.5M in THF) at room temperature under argon. The reaction mixture was stirred for 1 h and quenched with distilled water. The reaction mixture was partitioned between CH₂Cl₂ (10 mL) and water (15 mL), and the two layers separated. The organic layer was washed in succession with distilled water (2 × 10 mL) and saturated brine (2 × 10 mL), and dried over Na₂SO₄. Solvent was evaporated off to give 0.491 (86%) of 10l as a brownish solid. ¹H-NMR (CD₃OD, 400MHz) δ 3.05 (1H, s), 5.46 (1H, s), 7.66–7.78 (6H, m), 8.56 (2H, d, J = 9.2 Hz); HRMS (FAB, thioglycerol) calc for [C₁₄H₁₁NO + H]⁺ 210.0918, found 210.0909.

Representative Procedures for Diazo-transfer Reaction. Method A: p-Nitrobenzyl 6-azidopenicillanate (5a)

4-Nitrobenzyl 6-aminopenicillanate salt 3a (5.0 g, 9.5 mmol) was suspended in EtOAc (40 mL) and washed with saturated NaHCO₃ (2 × 30 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo to give pure 4-nitrobenzyl 6-aminopenicillanate. To a solution of 4-nitrobenzyl 6-aminopenicillanate in anhydrous CH₂Cl₂ (10 mL) was added triflyl azide solution (25 mmol) in CH₂Cl₂ (25 mL), and Et₃N (2.1 mL, 15.0 mmol). The reaction mixture was stirred at room temperature for 2 h. Solvent was evaporated off and the residue was directly purified by flash chromatography (silica, gradient 4:1; 3:1 Hexane/EtOAc) to give 2.35 g (68%) of 5a as a colorless gel. ¹H-NMR (CDCl₃, 400MHz) δ 1.38 (3H, s), 1.61 (3H, s), 4.49 (1H, s), 4.92 (1H, d, J = 4.0 Hz), 5.23 (2H, q, J =24.0, 12.0 Hz), 5.43 (1H, d, J = 4.0 Hz), 7.50 (2H, d, J = 8.8 Hz), 8.17 (2H, d, J = 8.8 Hz); ¹³C-NMR (CDCl₃, 100MHz) δ 26.4, 31.5, 64.3, 65.7, 66.5, 67.6, 70.0, 123.7, 128.0, 141.5, 147.9, 167.0, 169.8;

Method B: Benzhydryl 6-azidopenicillanate (5c)

Benzhydryl 6-aminopenicillanate salt 3c (3.5 g, 6.32 mmol) was suspended in EtOAc (60 mL) and washed with saturated NaHCO₃ (3 × 40 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo to give 2.4 g of pure benzhydryl 6-aminopenicillanate. To a solution of benzhydryl 6-aminopenicillanate in anhydrous CH₂Cl₂ (15 mL) was added triflyl azide solution (14 mmol) in CH₂Cl₂ (35 mL), DMAP (1.15 g, 9.24 mmol), and Et₃N (0.1 mL). The reaction mixture was stirred at room temperature for 1.5 h and solvent was evaporated off. The residue was directly purified by flash chromatography (silica, gradient 1:0; 5:1 Hexane/EtOAc) to give 1.38 g (54%) of 5c as a colorless gel. ¹H-NMR (CDCl₃, 400MHz) δ 1.26 (3H, s), 1.64 (3H, s), 4.57 (1H, s), 4.89 (1H, d, J = 4.0 Hz), 5.47 (1H, d, J = 4.0 Hz), 6.93 (1H, s), 7.28–7.35 (10H, m); ¹³C-NMR (CDCl₃, 100MHz) δ 26.2, 31.9, 64.7, 66.6, 67.8, 70.2, 78.4, 126.8, 127.5, 128.1, 128.4, 128.5, 138.8, 166.5, 169.8; HRMS (FAB, mnba) calc for [C₂₁H₂₀N₄O₃S + H]⁺ 409.1334, found 409.1357.

Method C: p-Nitrobenzyl 6-azidopenicillanate (5a)

4-Nitrobenzyl 6-aminopenicillanate salt 3a (0.75 g, 1.4 mmol) was suspended in EtOAc (30 mL) and washed with saturated NaHCO₃ (2 × 20 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo to give pure 4-nitrobenzyl 6-aminopenicillanate. To a solution of pure 4-nitrobenzyl 6-aminopenicillanate in anhydrous toluene (5 mL) was added triflyl azide solution (2.97 mmol) in toluene (25 mL), and Et₃N (0.3 mL, 2.1 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction was diluted with EtOAc (10 mL) and washed with 1N HCl (20 mL), distilled water (2 × 20 mL), and saturated brine (20 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo to give 361 mg (70%) of 5a as a yellowish solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.42 (3H, s), 1.66 (3H, s), 4.52 (1H, s), 4.92 (1H, d, J = 4.0 Hz), 5.26 (2H, q, J = 24.0, 12.0 Hz), 5.46 (1H, d, J = 4.0 Hz), 7.53 (2H, d, J = 8.8 Hz), 8.24 (2H, d, J = 8.8 Hz).

p-Methoxybenzyl 6-azidopenicillanate (5b)

6-azidopenicillanate 5b was prepared from p-Methoxybenzyl 6-aminopenicillanate 3b (4.0 g, 7.9 mmol) and triflyl azide (25 mmol) in CH₂Cl₂ (total volume = 25 mL), and Et₃N (1.44 mL, 10.3 mmol) using method A. The reaction mixture was stirred at room temperature for 2 h. Purification of the crude product by flash chromatography (silica, gradient 4:1; 3:1 Hexane/EtOAc) afforded 1.40 g (49%) of 5b as a colorless gel. ¹H-NMR (CDCl₃, 100MHz) δ 1.36 (3H, s), 1.61 (3H, s), 3.79 (3H, s), 4.45 (1H, s), 4.88 (1H, d, J = 4.0 Hz), 5.10 (2H, app. q, J = 24.0, 12.0 Hz), 5.44 (1H, d, J = 4.0 Hz), 6.87 (2H, d, J = 8.8 Hz), 7.27 (2H, d, J = 8.8 Hz); ¹³C-NMR (CDCl₃, 400MHz) δ 26.6, 31.7, 55.3, 64.6, 66.5, 67.4, 67.7, 70.2, 113.9, 126.5, 130.4, 159.7, 167.2, 169, 7; HRMS (FAB, mnba) calc for [C₁₆H₁₈N₄O₄S + H]⁺ 363.1127, found 363.1151.

Representative Procedure for Cu(I)-Catalyzed Cycloaddition Reaction. p-Methoxybenzyl benzyl-6-triazolylpenicillanate (8a)

p-Methoxybenzyl 6-azidopenicillanate 5b (0.27g, 0.635 mmol) and 3-phenyl-1-propyne 6 (0.17 mL, 1.39 mmol) were dissolved in anhydrous THF (8 mL) and stirred under argon at room temperature. Copper (I) iodide (0.011 g, 0.06 mmol), and Hunig’s base (0.1 mL) were then added to the reaction mixture, and stirring was continued for 2 h. The reaction mixture was diluted with CH₂Cl₂ (40 mL) and washed with 1:4 NH₄OH/saturated NH₄Cl (3 × 30 mL) and again with saturated NH₄Cl (30 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by flash chromatography (silica, gradient 3:1; 2:1; 3:2 Hexane/EtOAc) to give 246 mg (71%) of 8a as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.35 (3H, s), 1.57 (3H, s), 3.79 (3H, s), 4.08 (2H, s), 4.47 (1H, s), 5.12 (2H, app. q, J = 24.0, 12.0 Hz), 5.69 (1H, d, J = 4.0 Hz), 6.27 (1H, d, J = 4.4 Hz), 6.88 (2H, d, J = 8.8 Hz), 7.20–7.30 (7H, m), 7.42 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.9, 30.9, 32.0, 55.2, 65.3, 66.2, 67.3, 67.5, 70.5, 114.0, 122.4, 126.5, 128.5, 128.6, 130.6, 138.5, 147.2, 160.0, 167.1, 168.1; HRMS (FAB, thioglycerol) calc for [C₂₅H₂₆N₄O₄S + H]⁺ 479.1753, found 479.1756.

Benzhydryl benzyl-6-triazolylpenicillanate (8b)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.270 g, 0.661 mmol) and 3-phenyl-1-propyne 6 (0.17 mL, 1.39 mmol) within 2.5 h followed by flash chromatography (silica, gradient 3:1; 2:1; 3:2 Hexane/EtOAc) gave 246 mg (71%) of 8b as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.61 (3H, s), 1.57 (3H, s), 4.09 (2H, s), 4.59 (1H, s), 5.73 (1H, d, J = 4.0 Hz), 6.29 (1H, d, J = 4.4 Hz), 6.94 (1H, s), 7.21–7.35 (15H, m), 7.43 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 31.2, 32.0, 65.4, 66.3, 67.4, 70.6, 78.6, 122.4, 126.5, 126.9, 127.6, 128.3, 128.5, 128.6, 128.7, 138.5, 138.7, 138.8, 147.3, 166.3, 168.1; HRMS (FAB, mnba) calc for [C₃₀H₂₈N₄O₃S + H]⁺ 525.1956, found 525.1960.

Benzhydryl 2,6-dimethoxyphenyl-6-triazolylpenicillanate (8c)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.227 g, 0.555 mmol) and 2-Ethynyl-1,3-dimethoxybenzene 7 (0.06 g, 0.37 mmol) within 4 h followed by flash chromatography (silica gel, 1:1 Hexane/EtOAc) gave 120 mg (57%) of 8c as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.26 (3H, s), 1.69 (3H, s), 3.78 (6H, s), 4.64 (1H, s), 5.80 (1H, d, J = 4.8 Hz), 6.42 (1H, d, J = 4.4 Hz), 6.62 (2H, d, J = 8.4 Hz), 6.96 (1H, s), 7.26–7.35 (11H, m), 7.95 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 31.4, 56.0, 65.2, 66.4, 67.7, 70.6, 78.6, 104.1, 125.2, 126.9, 127.6, 128.3, 128.5, 128.6, 128.7, 129.9, 138.9, 139.9, 158.3, 166.4, 168.6; HRMS (FAB, thioglycerol) calcd for [C₃₁H₃₀N₄O₅S + H]⁺ 571.2015, found 571.2049.

Benzhydryl 4-pyridyl-6-triazolylpenicillanate (11a)

4-Ethynylpyridyl hydrochloride (0.06 g, 0.414 mmol) was suspended in EtOAc (30 mL) and washed with saturated NaHCO₃ (1 × 30 mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuo to give 4-ethynylpyridine 10a as white brown solid. The reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 10a (0.076 g, 0.734 mmol) within 3 h followed by flash chromatography (silica gel, gradient 1:1; 1:4 Hexane/EtOAc) gave 166 mg (74%) of 11a as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.26 (3H, s), 1.69 (3H, s), 4.65 (1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.39 (1H, d, J = 4.0 Hz), 6.96 (1H, s), 7.32–7.36 (10H, m), 7.77 (2H, d, J = 5.20 Hz), 8.19 (1H, s), 9.03(2H, d, J = 5.20 Hz); ¹³C-NMR (CDCl₃, 100MHz) δ 26.8, 31.1, 65.7, 66.2, 67.2, 70.8, 78.8, 120.1, 122.1, 126.7, 127.5, 128.2, 128.4, 128.5, 138.6, 138.6, 144.5, 149.2, 165.9, 167.5; HRMS (FAB, thioglycerol) calc for [C₂₈H₂₅N₅O₃S + H]⁺ 512.1756, found 512.1763.

Benzhydryl 3-pyridyl-6-triazolylpenicillanate (11b)

The reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.441 mmol) and 3-ethynylpyridine 10b (0.068 g, 0.661 mmol) within 3 h followed by flash chromatography (silica gel, gradient 1:1; 1:3 Hexane/EtOAc) gave 170 mg (76%) of 11b as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.26 (3H, s), 1.70 (3H, s), 4.66 (1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.40 (1H, d, J = 4.0 Hz), 6.96 (1H, s), 7.32–7.36 (10H, m), 8.13 (1H, s), 8.26 (1H, d, J = 8.24 Hz), 8.58 (1H, br s), 9.03(1H, br s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.9, 31.1, 65.7, 66.3, 67.3, 70.7, 78.7, 120.8, 126.7, 127.5, 128, 2, 128, 4, 128, 5, 133.8, 138.5, 138.6, 138.6, 144.0, 146.1, 148.3, 166.0, 167.6; HRMS (FAB, thioglycerol) calc for [C₂₈H₂₅N₅O₃S + H]⁺ 512.1756, found 512.1773.

Benzhydryl 2-pyridyl-6-triazolylpenicillanate (11c)

The reaction of benzhyldryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 2-ethynylpyridine 10c (0.06 mL, 0.55 mmol) within 6 h followed by flash chromatography (silica gel, gradient 4:1; 2:1; 1:2; Hexane/EtOAc) gave 145 mg (64%) of 11c as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.25 (3H, s), 1.71 (3H, s), 4.64 (1H, s), 5.78 (1H, d, J = 4.0 Hz), 6.39 (1H, d, J = 4.0 Hz), 6.96 (1H, s), 7.32–7.36 (10H, m), 7.78 (1H, m), 8.18 (1H, d, J = 7.69 Hz), 8.45 (1H, s), 8.58 (1H, d, J = 4.39 Hz); ¹³C-NMR (CDCl₃, 100MHz) δ 26.9, 31.3, 65.7, 66.5, 67.4, 70.8, 78.7, 120.5, 123.0, 126.8, 127.5, 128.2, 128.4, 128.5, 128.6, 138.6, 138.7, 166.1, 167.8; HRMS (FAB, thioglycerol) calc for [C₂₈H₂₅N₅O₃S + H]⁺ 512.1756, found 512.1765.

Benzhydryl 2-thiopyl-6-triazolylpenicillanate (11d)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 3-ethynylthiopene 10d (0.06 g, 0.55 mmol) within 5 h followed by flash chromatography (silica gel, gradient 1:0; 5:1; 3:1 Hexane/EtOAc) gave 150 mg (79%) of 11d as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.25 (3H, s), 1.69 (3H, s), 4.64 (1H, s), 5.78 (1H, d, J = 4.4 Hz), 6.36 (1H, d, J = 4.0 Hz), 6.96 (1H, s), 7.30–7.38 (11H, m), 7.44 (1H, dd, J =4.0, 1.2 Hz), 7.70 (1H, dd, J = 2.0, 1.2 Hz), 7.90 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 30.9, 66.1, 67.3, 70.8, 78.7, 120.1, 121.6, 125.7, 126.4, 126.9, 127.6, 128.3, 128.6, 128.7, 131.1, 138.7, 138.8, 143.8, 166.3, 168.2; HRMS (FAB, thioglycerol) calc for [C₂₇H₂₄N₄O₃S₂ + H]⁺ 517.1368, found 517.1374.

p-Methoxybenzyl phenyl-6-triazolylpenicillanate (11e)

Reaction of p-methoxybenzyl-6-azidopenicillanate 5b (0.15 g, 0.414 mmol) and phenylacetylene 10e (0.14 mL, 1.24 mmol) within 2 h followed by flash chromatography (silica gel, gradient 3:1; 3:2 Hexane/EtOAc) gave 165 mg (86%) of 11e as a white foam. ¹H-NMR (CDCl₃, 400MHz) δ 1.38 (3H, s), 1.66 (3H, s), 3.80 (3H, s), 4.54 (1H, s), 5.14 (2H, app. q, J = 24.0, 12.0 Hz), 5.75 (1H, d, J = 4.0 Hz), 6.37 (1H, d, J = 4.0 Hz), 6.89 (2H, d, J = 8.8 Hz), 7.29–7.35 (3H, m), 7.39–7.42 (2H, m), 7.82 (2H, m), 7.99 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 27.0, 30.8, 55.2, 65.4, 66.1, 67.2, 67.6, 70.7, 114.0, 120.3, 125.8, 126.5, 128.4, 128.8, 129.9, 130.6, 147.5, 160.0, 167.1, 168.2; HRMS (FAB, thioglycerol) calc for [C₂₄H₂₄N₄O₄S + H]⁺ 465.1596, found 465.1571.

Benzhydryl α-hydroxylbenzyl-6-triazolylpenicillanate (11f)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.2 g, 0.49 mmol) and 1-phenylprop-2-yn-1-ol 10f (0.13 g, 0.985 mmol) within 2 h followed by flash chromatography (silica gel, 2:1 Hexane/EtOAc) gave 191 mg (72%) of 11f as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.23 (3H, d, J = 12 Hz), 1.59 (3H, d, J = 8.0 Hz), 4.61 (1H, s), 5.71 (1H, d, J = 4.4 Hz), 5.99 (1H, d, J = 5.2 Hz), 6.23 (1H, dd, J = 4.0, 1.6 Hz), 6.95 (1H, s), 7.24–7.42 (15H, m), 7.57 (1H, s). ¹³C-NMR (CDCl₃, 100MHz) δ 26.6, 31.2, 31.3, 65.3, 65.3, 66.3, 66.4, 67.3, 67.4, 68.7, 68.9, 70.4, 70.5, 78.5, 122.0, 122.1, 126.1, 126.2, 126.3, 126.4, 126.6, 126.7, 127.1, 127.3, 127.6, 127.7, 128.0, 128.2, 128.3, 128.4, 138.5, 138.6, 141.5, 150.9, 166.0, 167.4, 167.5; HRMS (FAB, thioglycerol) calc for [C₃₀H₂₈N₄O₄S + H]⁺ 541.1909, found 541.1936.

Benzhydryl 4-biphenyl-6-triazolylpenicillanate (11g)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 4-ethynylbiphenyl 10g (0.098 g, 0.55 mmol) within 5 h followed by flash chromatography (silica gel, gradient 1:0; 5:1; 3:1 Hexane/EtOAc) gave 158 mg (73%) of 11g as a white solid. ¹H-NMR (DMSO-d₆, 400MHz) δ 1.26 (3H, s), 1.69 (3H, s), 4.92 (1H, s), 5.88 (1H, d, J = 2.0 Hz), 6.81 (1H, d, J = 2.0 Hz), 6.96 (1H, s), 7.32 (2H, d, J = 8.0 Hz), 7.38 (5H, t, J = 7.2 Hz), 7.47 (5H, t, J = 6.8 Hz), 7.52 (2H, d, J = 8.0 Hz), 7.77 (2H, d, J = 8.0 Hz), 7.90 (2H, d, J = 8.0 Hz), 8.66 (1H, s)

Benzhydryl 3-biphenyl-6-triazolylpenicillanate (11h)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 3-ethynylbiphenyl 10h (0.098 g, 0.55 mmol) within 5 h followed by flash chromatography (silica gel, gradient 1:0; 5:1; 3:1 Hexane/EtOAc) gave 215 mg (100%) of 11h as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.14 (3H, s), 1.58 (3H, s), 4.54 (1H, s), 5.68 (1H, d, J = 4.4 Hz), 6.28 (1H, d, J = 4.0 Hz), 6.86 (1H, s), 7.12 (1H, s), 7.19–7.24 (10H, m), 7.31–7.39 (3H, m), 7.45 (1H, d, J = 8.0 Hz), 7.52 (2H, d, J = 8.0 Hz), 7.69 (2H, d, J = 8.0 Hz), 7.95 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 30.9, 65.5, 66.2, 67.3, 70.7, 78.7, 120.5, 124.6, 124.7, 126.9, 127.2, 127.5, 127.6, 128.3, 128.5, 128.6, 128.7, 129.3, 130.4, 138.7, 138.8, 140.6, 141.9, 147.5, 166.3, 168.2; HRMS (FAB, thioglycerol) calc for [C₃₅H₃₀N₄O₃S + H]⁺ 587.2116, found 587.2143.

Benzhydryl 2-biphenyl-6-triazolylpenicillanate (11i)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 2-ethynylbiphenyl 10i (0.098 g, 0.55 mmol) within 5 h followed by flash chromatography (silica gel, gradient 1:0; 5:1; 3:1 Hexane/EtOAc) gave 150 mg (70%) of 11i as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.20 (3H, s), 1.47 (3H, s), 4.47 (1H, s), 5.63 (1H, d, J = 4.0 Hz), 6.26 (1H, d, J = 4.4 Hz), 6.80 (1H, s), 6.93 (1H, s), 7.22–7.45 (18H, m), 8.15 (1H, d, J = 8.0 Hz); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 31.3, 65.4, 66.3, 67.4, 70.4, 78.6, 123.0, 126.9, 127.2, 127.5, 127.8, 128.1, 128.2, 128.5, 128.6, 128.6, 128.7, 128.8, 129.2, 130.3, 138.8, 140.2, 141.5, 166.3, 167.0; HRMS (FAB, thioglycerol) calc for [C₃₅H₃₀N₄O₃S + H]⁺ 587.2116, found 587.2143.

Benzhydryl 6-methoxynapthalyl-6-triazolylpenicillanate (11j)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 2-ethynyl-6-methoxylnapthalene (0.067 g, 0.37 mmol) within 5 h followed by flash chromatography (silica gel, gradient 1:0; 5:1; 3:1 Hexane/EtOAc) gave 175 mg (81%) of 11j as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.27 (3H, s), 1.72 (3H, s), 3.91 (3H, s), 4.67 (1H, s), 5.80 (1H, d, J = 4.4 Hz), 6.41 (1H, d, J = 4.4 Hz), 6.97 (1H, s), 7.13–7.16 (2H, m), 7.31–7.35 (10H, m), 7.77 (2H, dd, J = 6.4, 2.8 Hz), 7.88 (1H, dd, J = 6.4, 1.6 Hz), 8.08 (1H, s), 8.26 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 31.0, 55.3, 65.5, 66.2, 67.3, 70.8, 78.7, 119.3, 120.2, 124.3, 124.6, 125.1, 126.9, 127.4, 127.6, 128.3, 128.6, 128.7, 128.9, 129.7, 134.4, 138.7, 138.8, 147.7, 158.0, 166.3, 168.2; HRMS (FAB, mnba) calc for [C₃₄H₃₀N₄O₄S + H]⁺ 591.2066, found 591.2061.

Benzhydryl 4-pyridylphenyl-6-triazolylpenicillanate (11k)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 4-(4-ethynylphenyl)pyridine 10k (0.098 g, 0.55 mmol) within 5 h followed by flash chromatography (silica gel, gradient 1:1; 1:2; 1:4 Hexane/EtOAc) gave 202 mg (94%) of 11k as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.27 (3H, s), 1.71 (3H, s), 4.66 (1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.40 (1H, d, J = 4.0 Hz), 6.97 (1H, s), 7.31–7.35 (12H, m), 7.61 (2H, br s), 7.72 (2H, d, J = 8.0 Hz), 7.97 (2H, d, J = 8.0 Hz), 8.09 (1H, s); ¹³C-NMR (CDCl₃, 100MHz) δ 26.7, 30.9, 65.6, 66.2, 67.3, 70.7, 78.7, 120.8, 126.5, 126.9, 127.5, 127.6, 128.3, 128.6, 128.7, 131.2, 137.3, 138.7, 138.8, 146.6, 148.8, 148.9166.3, 168.1; HRMS (FAB, thioglycerol) calc for [C₃₄H₂₉N₅O₃S + H]⁺ 588.2069, found 588.2100.

Benzhydryl 4-pyridylbenzylhydroxyl-6-triazolylpenicillanate (11l)

Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g, 0.367 mmol) and 1-(4-(pyrdin-4-yl)prop-2-yn-1-ol 10l (0.115 g, 0.55 mmol) within 2 h followed by flash chromatography (silica gel, gradient 1:2; 1:3 Hexane/EtOAc) gave 190 mg (84%) of 11l as a white solid. ¹H-NMR (CDCl₃, 400MHz) δ 1.22 (3H, d, J = 12 Hz), 1.60 (3H, d, J = 5.6 Hz), 4.58 (1H, s), 5.72 (1H, d, J = 4.4 Hz), 6.08 (1H, s), 6.28 (1H, d, J = 4.4 Hz), 6.92 (1H, s), 7.24–7.32 (10H, m), 7.44 (2H, dd, J = 6.0, 1.6 Hz) 7.52–7.58 (4H, m), 7.67 (1H, s), 8.53 (2H, dd, J = 6.4, 1.6 Hz). ¹³C-NMR (CDCl₃, 100MHz) δ26.7, 30.6, 31.1, 31.2, 64.3, 65.4, 66.2, 66.3, 67.3, 68.4, 68.5, 70.5, 78.6, 121.4, 122.0, 126.6, 126.9, 127.1, 127.4, 128.1, 128.3, 128.4, 128.5, 137.1, 137.2, 138.5, 142.9, 147.8, 149.6, 150.8, 150.9, 166.0, 167.5, 167.6; HRMS (FAB, thioglycerol) calc for [C₃₅H₃₁N₅O₄S + H]⁺ 618.2175, found 618.2180

Representative Procedure for Deprotection of Carboxyl Protecting Group. Benzyl-6-triazolylpenicillanic acid (9a)

p-Methoxybenzyl benzyl-6-triazolylpenicillanate 8a (0.13 g, 0.26 mmol) was dissolved and stirred in anhydrous CH₂Cl₂ (1 mL) at −5°C. Anhydrous anisole (0.2 mL, 1.83 mmol) and trifluoroacetic acid (0.5 mL, 6.49 mmol) were added, and the reaction mixture stirred for 2 h. The reaction mixture was diluted with cold Et₂O (10 mL), and the solvent was evaporated off in an ice bath. The residue was concentrated in vacuo on an ice bath for 15 minutes, and redissolved in THF (5 mL) and ½ saturated NaHCO₃ (15 mL) at 0°C. The resulting mixture was stirred at 0°C for 15 minutes and partitioned between deionized water (5 mL) and EtOAc (20 mL). The two layers were separated and the aqueous layer was extracted with EtOAc (2 × 20 mL). The aqueous layer was acidified to pH 3 in an ice bath with 1N HCl and extracted with EtOAc (3 × 20 mL). The combined organic layer was dried over Na₂SO₄ and concentrated in vacuo to give 89 mg (100%) of 9a as a white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.55 (3H, s), 1.70 (3H, s), 4.07 (2H, s), 4.55 (1H, s), 5.85 (1H, d, J = 4.4 Hz), 6.58 (1H, d, J = 4.4 Hz), 7.26–7.28 (5H, m), 7.79 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 26.8, 31.0, 31.8, 65.1, 67.0, 67.8, 70.7, 123.2, 126.7, 128.8, 128.9, 129.0, 139.9, 168.4, 168.5; HRMS (FAB, mnba) calc for [C₁₇H₁₈N₄O₃S + H]⁺ 359.1177, found 359.1177.

2,6-Dimethoxyphenyl-6-triazolylpenicillanic acid (9b)

The reaction of a CH₂Cl₂ solution of 8c with anisole and TFA within 2 h, as described for the synthesis of 9a from 8a, afforded 85 mg (100%) of 9b as a white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.59 (3H, s), 1.77 (3H, s), 3.77 (6H, s), 4.62 (1H, s), 5.93 (1H, d, J = 4.4 Hz), 6.73 (1H, d, J = 4.4 Hz), 6.74 (2H, d, J = 8.4 Hz), 7.35 (1H, t, J = 8.4 Hz), 8.15 (1H, s); ¹³C-NMR (acetone-d₆, 400MHz) δ 26.9, 31.2, 55.9, 65.2, 67.2, 68.0, 70.7, 104.6, 126.2, 131.0, 158.7, 168.6; HRMS (FAB, mnba) calc for [C₁₈H₂₀N₄O₅S + H]⁺ 405.1232, found 405.1214.

4-pyridyl-6-triazolylpenicillanic acid (12a)

The reaction of a CH₂Cl₂ solution of 11a with anisole and TFA within 0.5 h, as described for the synthesis of 9a from 8a, afforded 80 mg (100%) of 12a as a white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.56 (3H, s), 1.73 (3H, s), 4.60 (1H, s), 5.95 (1H, d, J = 4.0 Hz), 6.78 (1H, d, J = 4.0 Hz), 8.57 (2H, br s), 9.01 (2H, br s), 9.07 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 27.1, 31.6, 65.7, 67.7, 68.0, 71.4, 122.8, 126.8, 142.8, 143.6, 146.6, 167.5, 170.7; HRMS (FAB, mnba) calc for C₁₅H₁₅N₅O₃S + H]⁺ 346.0973, found 346.0972.

3-pyridyl-6-triazolylpenicillanic acid (12b)

The reaction of a CH₂Cl₂ solution of 11b with anisole and TFA within 0.5 h, as described for the synthesis of 9a from 8a, afforded 80 mg (100%) of 12b as a reddish white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.57 (3H, s), 1.74 (3H, s), 4.60 (1H, s), 5.95 (1H, d, J = 4.0 Hz), 6.76 (1H, d, J = 4.0 Hz), 8.15 (1H, br s), 8.84 (1H, s), 8.92 (2H, m), 9.41 (1H, br s); HRMS (FAB, mnba) calc for [C₁₅H₁₅N₅O₃S + H]⁺ 346.0973, found 346.0972.

2-pyridyl-6-triazolylpenicillanic acid (12c)

The reaction of a CH₂Cl₂ solution of 11c with anisole and TFA within 0.5 h, as described for the synthesis of 9a from 8a, afforded 80 mg (100%) of 12c as a white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.57 (3H, s), 1.76 (3H, s), 4.63 (1H, s), 5.95 (1H, d, J = 4.0 Hz), 6.79 (1H, d, J = 4.0 Hz), 7.79 (1H, m), 8.39 (1H, m), 8.48 (1H, m), 8.90 (1H, d, J = 5.1 Hz), 8.95 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 27.1, 31.5, 65.7, 67.6, 68.0, 71.2, 122.8, 125.3, 125.7, 143.2, 145.9, 146.7, 160.2, 167.8, 168.6; HRMS (FAB, mnba) calc for [C₁₅H₁₅N₅O₃S + H]⁺ 346.0973, found 346.0974.

2-Thiopyl-6-triazolylpenicillanic acid (12d)

The reaction of a CH₂Cl₂ solution of 11d with anisole and TFA within 1 h, as described for the synthesis of 9a from 8a, afforded 81 mg (100%) of 12d as a yellowish solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.57 (3H, s), 1.74 (3H, s), 4.59 (1H, s), 5.90 (1H, d, J = 4.0 Hz), 6.65 (1H, d, J = 4.0 Hz), 7.54 (1H, m), 7.57 (1H, dd, J = 3.0, 1.5 Hz), 7.87 (1H, dd, J = 3.0, 1.5 Hz), 8.34 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 27.1, 31.3, 65.4, 67.3, 68.0, 71.2, 121.4, 121.6, 126.3, 127.1, 132.4, 143.7, 168.3, 168.9; HRMS (FAB, mnba) calc for [C₁₄H₁₄N₄O₃S₂ +H]⁺ 351.0585, found 351.0577.

Phenyl-6-triazolylpenicillanic acid (12e)

The reaction of a CH₂Cl₂ solution of 11e with anisole and TFA within 2 h, as described for the synthesis of 9a from 8a, afforded 68.5 mg (93%) of 12e as a white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.47 (3H, s), 1.67 (3H, s), 4.54 (1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.76 (1H, d, J = 4.4 Hz), 7.34 (1H, m), 7.44 (2H, m), 7.89 (2H, m), 8.60 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 27.2, 31.2, 65.4, 67.3, 68.0, 71.2, 121.6, 126.1, 126.1, 128.6, 129.4, 131.3, 147.3, 168.4; HRMS (FAB, mnba) calc for [C₁₆H₁₆N₄O₃S + H]⁺ 345.1021, found 345.1015.

α-Hydroxylbenzyyl-6-triazolylpenicillanic acid (12f)

The reaction of a CH₂Cl₂ solution of 11f with thioanisole and TFA within 0.5 h, as described for the synthesis of 9a from 8a, afforded 76 mg (100%) of 12f as a pale yellowish solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.53 (3H, s), 1.67 (3H, s), 3.37 (1H, s), 4.55 (1H, s), 5.84 (1H, bs), 5.97 (1H, s), 6.55 (1H, bs), 7.22–7.43 (4H, m), 7.80 (1H, s); HRMS (FAB, thioglycerol) calc for [C₁₇H₁₈N₄O₄S + H]⁺ 375.1127, found 375.1192.

4-Biphenyl-6-triazolylpenicillanic acid (12g)

The reaction of a CH₂Cl₂ solution of 11g with anisole and TFA within 2 h, as described for the synthesis of 9a from 8a, afforded 65 mg (100%) of 12g as a yellowish solid. ¹H-NMR (DMSO-d₆, 400MHz) δ 1.48 (3H, s), 1.65 (3H, s), 4.38 (1H, s), 5.79 (1H, d, J = 4.0 Hz), 6.72 (1H, d, J = 4.0 Hz), 7.35–7.40 (1H, m), 7.47 (2H, t, J = 8.0 Hz), 7.74 (4H, dd, J = 11.2, 8.0 Hz), 7.99 (2H, d, J = 8.0 Hz), 8.63 (1H, s); HRMS (FAB, mnba) calc for [C₂₂H₂₀N₄O₃S + H]⁺ 421.1334, found 421.1333.

3-Biphenyl-6-triazolylpenicillanic acid (12h)

The reaction of a CH₂Cl₂ solution of 11h with anisole and TFA within 2 h, as described for the synthesis of 9a from 8a, afforded 54 mg (83%) of 12h as a white solid. ¹H-NMR (acetone-d₆, 400MHz) δ 1.59 (3H, s), 1.77 (3H, s), 4.61 (1H, s), 5.93 (1H, d, J = 4.0 Hz), 6.71 (1H, d, J = 4.0 Hz), 7.38 (1H, m), δ 7.48 (2H, m), 7.54 (1H, t, J = 8.0 Hz), 7.64 (1H, m), 7.72 (2H, m), 7.96 (1H, dt, J = 8.0, 1.5 Hz), 8.23 (1H, t, 1.5 Hz), 8.58 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 27.2, 31.2, 65.5, 67.3, 68.0, 71.3, 122.0, 124.6, 125.1, 127.2, 127.5, 128.1, 129.4, 130.0, 131.9, 141.1, 142.2, 147.2, 168.4, 168.9; HRMS (ESI, mnba) calc for [C₂₂H₂₀N₄O₃S + H]⁺ 421.1329, found 421.1312.

2-Biphenyl-6-triazolylpenicillanic acid (12i)

The reaction of a CH₂Cl₂ solution of 11i with anisole and TFA within 1 h, as described for the synthesis of 9a from 8a, afforded 65 mg (100%) of 12i as a white solid. H-NMR (acetone-d₆, 400MHz) δ 1.53 (3H, s), 1.59 (3H, s), 4.41 (1H, s), 5.78 (1H, d, J = 4.0 Hz), 6.55 (1H, d, J = 4.0 Hz), 7.00 (1H, s), 7.22–7.25 (2H, m), 7.32–7.52 (6H, m), 8.07 (1H, dd, J = 5.8, 1.5 Hz); HRMS (ESI, mnba) calc for [C₂₂H₂₀N₄O₃S + H}⁺ 421.1329, found 421.1359.

6-Methoxynaphthalyl-6-triazolylpenicillanic acid (12j)

The reaction of a CH₂Cl₂ solution of 11j with anisole and TFA within 1 h, as described for the synthesis of 9a from 8a, afforded 56 mg (89%) of 12j as a white foam. ¹H-NMR (DMSO-d₆, 400MHz) δ 1.58 (3H, s), 1.78 (3H, s), 3.91 (3H, s), 4.17 (1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.62 (1H, d, J = 4.0 Hz), 7.16 (1H, dd, J = 6.5, 2.2 Hz), 7.30 (1H, m), 7.85 (2H, dd, J = 3.0, 5.0 Hz), 7.98 (1H, m), 8.39 (1H, m), 8.55 (1H, s); ¹³C-NMR (acetone-d₆, 100MHz) δ 26.7, 30.7, 55.1, 65.0, 67.0, 67.6, 70.8, 106.1, 119.5, 121.3, 124.4, 124.5, 126.2, 127.7, 129.3, 129.9, 134.9, 147.4, 158.4, 168.3, 168.4; HRMS (FAB, thioglycerol) calc for [C₂₁H₂₀N₄O₄S + H]⁺ 425.1283, found 425.1264.

4-Pyridylphenyl-6-triazolylpenicillanic acid (12k)

The reaction of a CH₂Cl₂ solution of 11k with anisole and TFA within 1 h, as described for the synthesis of 9a from 8a, afforded 37 mg (65%) of 12k as a white foam. ¹H-NMR (acetone-d₆, 400MHz) δ 1.58 (3H, s), 1.77 (3H, s), 4.61 (1H, s), 5.94 (1H, d, J = 4.0 Hz), 6.74 (1H, d, J = 4.0 Hz), 8.17 (4H, m), 8.47 (2H, br s), 8.64 (1H, s), 9.04 (2H, br s); HRMS (FAB, mnba) calc for [C₂₁H₁₉N₅O₃S + H]⁺ 422.1287, found 422.1301.

4-Pyridylbenzylhydroxyl-6-triazolylpenicillanic acid salt (12l)

Benzhydryl 4-pyridylphenyl-6-triazolylpenicillanate 11l (0.09 g, 0.14 mmol) was dissolved and stirred in anhydrous CH₂Cl₂ (1 mL) at −5°C. Anhydrous thioanisole (0.2 mL, 1.83 mmol) and TFA (0.5 mL, 6.49 mmol) were added, and the reaction mixture stirred for 0.5 h. The reaction mixture was diluted with cold Et₂O (10 mL), and the solvent was evaporated off in an ice bath. The residue was concentrated in vacuo on an ice bath for 15 minutes, and redissolved in aqueous NaHCO₃ (0.018 g, 0.21 mmol) at 0°C. The resulting mixture was stirred at 0°C for 15 minutes and partitioned between deionized water (5 mL) and EtOAc (20 mL). The two layers were separated and the aqueous layer was extracted with EtOAc (2 × 20 mL). The aqueous layer was frozen in acetone-dry ice bath and concentrated in vacuo to give 35 mg (52%) of 12l as a white solid. ¹H-NMR (DMSO-d₆, 400MHz) δ 1.46 (3H, s), 1.61 (3H, m), 4.51 (1H, s), 5.75 (1H, d, J = 4.4 Hz), 5.94 (1H, s), 6.67 (1H, d, J = 4.0 Hz), 7.57 (2H, d, J = 8.0 Hz), 7.86 (4H, m), 8.75 (2H, m); HRMS (FAB, mnba) calc for [C₂₂H₂₀N₅NaO₄S + H]⁺ 474.1212, found 474.1234.

Figure 2.

The structure of some acid- and β-lactamase-resistant penicillins.