Routes of Synthesis of Carbapenems for Optimizing Both the

Article pubs.acs.org/jmc

Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D‑Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by β‑Lactamase BlaC Laura Iannazzo,† Daria Soroka,‡ Sébastien Triboulet,‡ Matthieu Fonvielle,⊥ Fabrice Compain,‡ Vincent Dubée,‡,§ Jean-Luc Mainardi,‡,¶ Jean-Emmanuel Hugonnet,‡ Emmanuelle Braud,† Michel Arthur,*,⊥,# and Mélanie Etheve-Quelquejeu*,†,# †

Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, team CBNIT, Université Paris Descartes, CNRS UMR 8601, Paris F-75006, France ‡ UPMC Univ Paris 06, LRMA, Equipe 12 and ⊥INSERM and Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Sorbonne Universités, Paris F-75006, France § Assistance Publique-Hôpitaux de Paris, Service de Réanimation Médicale, Hôpital Saint-Antoine, Paris F-75012,France ¶ Assistance Publique-Hôpitaux de Paris, Service de Microbiologie, Hôpital Européen Georges Pompidou, Paris F-75015, France S Supporting Information *

ABSTRACT: Combinations of β-lactams of the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for the treatment of drugresistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual β-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum β-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the β-lactam and the fivemembered rings of the carbapenem core. In particular, we show that the azide−alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable β-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.

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ing to the carbapenem class (Figure 1A),8 was found to be uniformly active against drug-resistant M. tuberculosis.9 Combinations of carbapenems with clavulanate have also been reported to be active in mouse models of infections that reproduce human pharmacokinetics10 and to achieve bacterial killing in macrophages.11 Furthermore, the combinations were superior to the first line drug isoniazide in eliminating a subpopulation of nongrowing but metabolically active cells believed to be responsible for relapses.11 These observations have stimulated clinical trials12 to evaluate the early bactericidal activity of these combinations, and the results of the trials are pending. The mode of action of β-lactams against M. tuberculosis is complex. First, the drug must escape hydrolysis by BlaC to reach its targets, which catalyze the last synthesis step of the cell wall peptidoglycan.5 This is thought to be conditioned by the

INTRODUCTION Tuberculosis (TB) remains a major medical problem with estimated numbers of new cases and deaths of 9.6 and 1.2 million in 2014, respectively.1 Worldwide, the TB mortality and prevalence rates fell by 47 and 42% since 1990. However, drugresistant TB (DR-TB) threatens global TB control and incurs use of less efficient and more toxic drugs for longer time periods with poorer treatment outcomes. The therapeutic options for DR-TB remain too limited in spite of intensive research for new drugs that recently led to the emergence of bedaquiline2 and delamanid.3 An alternative approach relies on the repurposing of ancient drugs that are not part of the recommended treatment of TB, such as the β-lactams.4 These drugs are considered as inefficient for the treatment of TB since Mycobacterium tuberculosis produces a broad spectrum class A β-lactamase (BlaC).5 However, the interest for β-lactams has been renewed by the observation that clavulanate, a broadly used β-lactamase inhibitor,6 irreversibly inactivates BlaC.7 In combination with clavulanate, meropenem, a β-lactam belong© XXXX American Chemical Society

Received: January 20, 2016

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DOI: 10.1021/acs.jmedchem.6b00096 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structure of carbapenems. (A) Carbapenems approved for human use. Related compounds, imipenem and faropenem, were not considered in this study since they contain different five-membered rings.8 (B) Target compounds and structural analogy between our triazole carbapenems and the side chain of ertapenem. Dashes highlight the position of the chemical modifications. The triazole ring recapitulates the charge distribution of an amide bond.28 (C) Synthetic routes developed in this study from compound 3.

relative velocity of target inactivation and drug hydrolysis.13 Poor substrates of BlaC, such as meropenem, are of particular interest since residual β-lactamase activity, which may persist in vivo in spite of treatment with clavulanate, is expected to be insignificant if the carbapenem hydrolysis efficiency is low.9 Furthermore, very poor substrates of BlaC might be used in the absence of a β-lactamase inhibitor allowing simplifying therapeutic regimens.11 This could also be crucial to prevent emergence of inhibitor-resistant forms of BlaC, which, to date, have only been documented based on kinetic analyses of purified enzymes obtained by site-directed mutagenesis.14−16 In M. tuberculosis, the targets of β-lactams are unusually diverse due to the presence of two modes of synthesis of cell wall peptidoglycan.17,18 This polymer consists of glycan chains made of alternate N-acetylglucosaminyl (GlcNAc) and Nglycolylmuramyl (MurNGlyc) residues, the latter aminosugar being substituted by a short stem peptide made by sequential addition of L-Ala1, D-Glu2, which is secondarily amidated (DisoGln), mesodiaminopimelic acid3 (DAP), and of the dipeptide D-Ala4-D-Ala5. In the pentapeptide, DAP3 is linked by its L-center to the γ-carboxyl of D-isoGln2.19 Stem peptides carried by adjacent glycan chains are linked together by amide

bonds. In most bacteria, the cross-linking reaction involves cleavage of the D-Ala4-D-Ala5 peptide bond of an acyl donor and linkage of the carbonyl of D-Ala4 to the side chain amino group carried by the diamino acid at the third position of the acyl acceptor (located at the D-center of DAP3 in M. tuberculosis).20 The resulting D-Ala4 → DAP3 cross-links are formed by D,Dtranspeptidases that are the only essential targets of β-lactam antibiotics in most bacteria. In M. tuberculosis, this mode of peptidoglycan synthesis is functional but only accounts for a minority (ca. 20%) of the cross-links present in mature peptidoglycan.18 The majority of the cross-links is formed by 3 4 L,D-transpeptidases (Ldts), which cleave the DAP -D-Ala bond of the donor and generate DAP3 → DAP3 cross-links.18,21 Since Ldts exclusively use acyl donors containing a stem tetrapeptide, this mode of cross-linking requires D,D-carboxypeptidases that cleave the D-Ala4-D-Ala5 peptide bond of stempentapeptides.21,22 The D,D-carboxypeptidases are dispensable in most bacteria but are likely to be essential targets of β-lactams in M. tuberculosis due to their roles in generating the substrates of Ldts.23 The β-lactams are structure analogues of the D-Ala4-D-Ala5 extremity of peptidoglycan precursors.24 The drugs act as B



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Scheme 1. Synthesis of Alkyne Carbapenem 4a

a

Reaction conditions: (a) i. NaOH 1M, DMF, ii. DOWEX H+; (b) DIPEA, DMF 0 to 5 °C, 20 h.

Figure 2. Structure of azides 5a−h and of triazole-containing carbapenems 6a−h and 7a−h. The asterisk (*) denotes that compounds 7d and 7g could not be evaluated in biological tests due to limited availability and instability in solution, respectively.

therapy of TB, carbapenems have never been optimized for inactivation of the unusual β-lactam targets of M. tuberculosis or to limit their detoxification by the β-lactamase BlaC, which displays moderate but significant carbapenemase activity.5,7,9 Here, we report three routes of carbapenem synthesis for versatile access to modifications of their side chains. Biological evaluation of the new carbapenems is based on kinetic analyses of their hydrolysis by the β-lactamase BlaC and their capacity to irreversibly inactivate the L,D-transpeptidase LdtMt1, a representative of the five paralogs of M. tuberculosis.18,27 We show that the side chains of carbapenems can be modified to improve both drug stability and target inactivation.

suicide substrates leading to irreversible acylation of the activesite serine of D,D-transpeptidases. Ldts were unexpectedly found to be similarly inactivated by acylation of their active-site cysteine in spite of the difference in the stereospecificity of the two types of transpeptidases, which involves cleavage of an L-D (DAP3-D-Ala4) instead of a D-D (D-Ala4-D-Ala5) peptide bond.25 However, the inactivation of Ldts is efficient only with a single class of β-lactams, the carbapenems.25 Consequently, crosslinking by Ldts is potentially associated with high-level resistance to other classes of β-lactams, such as the cephems (cephalosporins) and the penams (penicillins), both due to slow acylation and instability of the acylenzyme.26 Carbapenems have been developed and optimized for the treatment of infections due to Gram-negative bacteria producing extended-spectrum β-lactamases active on thirdgeneration cephalosporins.8 As a consequence of the limited efforts previously made for the development of β-lactam-based

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RESULTS AND DISCUSSION Routes of Synthesis of Carbapenems. To obtain new carbapenems, we chose to modify the side chain of the fivemembered ring of the carbapenem core and to functionalize the C



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Scheme 2. CuI-Catalyzed Cycloaddition of Azide Compounds to the Alkyne Carbapenem and Deprotection of the Carboxylic Acida

a

(a) Condition A: CuSO4 30 mol %, sodium ascorbate 60 mol %, DMF/H2O (2/1), rt, overnight, condition B: CuTC 30 mol %, toluene, rt, 8 h; (b) H2, 3.5 bar, Pd/C 10% 3 equiv, 2 h, THF/triethylammonium-bicarbonate (pH 8.5) (1/1).

Scheme 3. Synthesis of Carbapenems 9a−c and 10a−c by a Direct Addition of a Sulfhydryl Compounda

a Reaction conditions: (a) DIPEA, MeCN, 0−5 °C, 20 h; (b) H2, 3.5 bar Pd/C 10% 3 equiv, 2 h, THF/triethylammonium-bicarbonate (pH 8.5) (1/ 1).

Figure 3. Structure of sulfhydryl 8a−c and of carbapenems 9a−c and 10a−c.

hydroxyl group of its β-lactam ring (Figure 1B,C). We use two approaches to modify the side chain of the five-membered ring. The first approach is based on copper(I)-catalyzed 1,3 cycloaddition (CuAAC) between the alkyne 4 and various azide compounds (Figure 1C; Route 1). The CuAAC reaction has been investigated since it is performed under mild conditions and is compatible with diverse functional groups.28−30 We have therefore anticipated that it might also be compatible with the highly unstable β-lactam ring of carbapenems. All compounds obtained by this approach contain a triazole ring that mimics features of clinically used carbapenems (Figure 1B). In order to generate additional diversity in the side chain of the five-membered ring, we use a second approach involving the direct addition of various sulfhydryl compounds to the activated carbapenem 3 (Figure 1C; Route 2). This approach also enables evaluation of the impact of the triazole ring on the biological activities. In order to modify the side chain of the β-lactam ring, we have also developed esterification of the C8 hydroxyl (Figure 1C; Route 3). Copper(I)-Catalyzed 1,3 Cycloaddition. We first synthesized in 66% yield alkynyl carbapenem 4 by addition of butynthiol 2 to β-methyl vinylphosphate 3 in the presence of DIPEA in DMF (Scheme 1). Butynthiol 2 was generated in situ from 3-butynyl ethanethioate 1. Then, we focused on the access to azide partners 5a−h (Figure 2). Briefly, compounds 5a and

5g were commercially available. Compounds 5b−d were obtained by protecting the carboxylic acids of the respective precursors with a para-nitrobenzyl group (PNB) (see Supporting Information). Compounds 5e, 5f, and 5h were prepared by substitution of bromine by an azido group, followed for 5e and 5f by protection of carboxylic acids with PNB (see Supporting Information). Two conditions (A and B) have been used for the CuAAC reaction (Scheme 2). Azides 5a−e, containing an unsubstituted phenyl or a phenyl substituted in the ortho, meta, and para positions by a nitrobenzyl ester, were subjected to condition A using CuSO4 (30 mol %) and sodium ascorbate (60 mol %) in DMF/H2O overnight at room temperature. This condition led to the functionalized carbapenems 6a−e in 43−60% yield. Condition A was also used for compounds 5f and 5h, bearing a carboxyl on an alkyl chain and an indole group, respectively, to obtain triazole derivatives 6f and 6h in 46 and 71% yield. To obtain the sulphonyl triazole 6g from thesulfonyl azides 5g, a room-temperature method was used in the presence of CuI thiophene carboxylate CuTC (30 mol %) in toluene at room temperature for 8 h (condition B).31 This reaction afforded the triazole 6g in 37% yield. Deprotection of the carboxylic groups of carbapenems 6a−h was performedby hydrogenolysis (Pd/C; 3.5 bar H2) under biphasic conditions (THF/triethylammonium-bicabonate; pH 8.5) to provide carbapenems 7a−h in 10−26% yield. D



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Scheme 4. Synthesis of Carbapenems 12a and 12b and 13a and 13ba

Reaction conditions: (a) DMAP, NEt3, DCM, 0 °C to rt, overnight; (b) H2, 3.5 bar Pd/C 10% 3 equiv, 2 h, THF/triethylammonium-bicarbonate (pH 8.5) (1/1).

a

Figure 4. Structure of acyl chlorides 11a and 11b and of carbapenems 12a and 12b and 13a and 13b.

Direct Addition of Sulfhydryl Compounds. We have recently reported addition of a sulfhydryl compound 8a to βmethyl vinylphosphate 3 to obtain the protected carbapenem 9a (Scheme 3).32 The same approach was used for addition of para-nitrobenzyloxycarbonyl cysteamine 8b33 and the commercially available 2-phenylethanethiol 8c to 3 affording protected carbapenem 9b and 9c in 74 and 83% yield, respectively (Figure 3). The final deprotection step was performed as described above leading to carbapenem 10a−c in 20 to 40% yield. Esterification of the C8 Hydroxyl of the β-Lactam Ring. For this approach, we used protected carbapenem 9a (above) containing an ethyl side chain on the five-membered carbapenem ring (Scheme 4). Compound 9a in the presence of the acyl chlorides 11a and 11b, afforded acylated compounds 12a and 12b in 63 and 81% yield, respectively (Figure 4). Deprotection led to carbapenems 13a and 13b in 30 and 10% yield, respectively. Inactivation of the L,D-Transpeptidase LdtMt1 by Carbapenems. Analysis of the M. tuberculosis genome has previously revealed five putative L,D-transpeptidases that have been characterized with respect to their inactivation by carbapenems approved for use in humans and their capacity to catalyze formation of DAP3 → DAP3 cross-links in vitro.27,34 In this study, we focus on one of these enzymes, LdtMt1, which has been previously characterized in details at the structural and functional levels.34−36 Inactivation of LdtMt1 by carbapenems is a two-step reaction. In the first step, nucleophilic attack of the carbon of the β-lactam carbonyl by the sulfur of the catalytic cysteine leads to reversible formation of an oxyanion.26,37,38 The second step is irreversible and involves rupture of the βlactam ring C−N bond and protonation of the nitrogen. Spectrofluorimetry has been used to determine the second- and first-order constant k1 and k2 for the formation of the oxyanion and the acylenzyme, respectively.26,37 The kinetic parameter k2/ Kapp is used to evaluate the overall efficiency of the reaction.26,34 Kinetic parameters k1, k2, and k2/Kapp for acylation of LdtMt1 by carbapenems 7a−h, 10a−c, and 13a−b were determined

(Table 1). Kinetic parameters determined for three carbapenems approved for use in humans, meropenem, ertapenem, and doripenem, have been included for comparison. These analyses revealed the following structure−activity relationships. The triazole ring present in carbapenems 7a−h is expected to mimic the amide present in meropenem and ertapenem (Figure 1B). Mass spectrometry analyses indicated that the triazole linker was stable within the active site of LdtMt1, as shown by detection of covalent adducts resulting from the acylation of the active-site cysteine by the carbapenems (data not shown). Comparison of compounds 7a and 10c, containing a phenyl linked to the carbapenem core by a triazole or an alkyl chain, respectively, indicated that the triazole is well tolerated for efficient inactivation of LdtMt1 (Table 1). Introduction of a carboxyl onto the triazole-linked phenyl group was explored to enhance the solubility of the final deprotected carbapenems in aqueous media (compounds 7b− e) and to mimic the structure of ertapenem (Figure 1B). However, compounds 7a and 7h, containing an unsubstituted phenyl and an indole group, respectively, were soluble in phosphate buffer (pH 6.4) up to the highest tested concentration (1000 μg/mL), indicating that the carboxyl group is dispensable for solubility. Kinetic parameters k1, k2, and k2/Kapp were not greater for carbapenems containing a phenyl substituted by a carboxyl group (7b and 7c) than for 7a, which harbors an unsubstituted phenyl (Table 1). These results indicate that the carboxylic group, also present in ertapenem, is unlikely to contribute to velocity of the inactivation reaction. The impact of the carboxylic acid position on the kinetics of LdtMt1 inactivation was tested for the ortho and meta positions (7b and 7c). We also synthesized 7f, which carries the carboxyl group at the extremity of a butyl chain. None of these compounds showed very important differences with respect to the kinetic parameters for the reaction of acylation of LdtMt1 (Table 1). The presence of the carboxyl on the butyl chain (7f) was the least favorable with overall inactivation efficiencies (k2/ Kapp) ca. 3-fold lower than those observed for the other compounds. Introduction of a linker between the phenyl and E



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Table 1. Kinetic Parameters for Inactivation of the L,D-Transpeptidase LdtMt1 and Hydrolysis by the β-Lactamase BlaCa

a

M, meropenem; E, ertapenem; D, doripenem.

hydroxyl group of the β-lactam side chain. This result is surprising since this group is in hydrogen interaction with the hydroxyl of Tyr190 in the crystal structure of LdtMt1 acylated by imipenem.35,36 In conclusion, our data indicate that a wide variety of chemical groups can be introduced in both side chains of carbapenems without impairing the efficiency of the acylation reaction. However, none of the side chains provided strong improvement of kinetic parameters in comparison to 10a containing a minimal side chain. Of note, the efficiency of the acylation reaction (k2/Kapp) was 2−26-fold greater for our carbapenems than for meropenem and doripenem, suggesting that the side chain of the latter drugs may have a negative impact. Hydrolysis of Carbapenems by β-Lactamase BlaC from M. tuberculosis. Kinetic parameters kcat, Km, and kcat/ Km for hydrolysis of carbapenems 7a−h, 10a−c, and 13a and

the triazole ring (7e) did not improve inactivation of LdtMt1 in comparison to 7b and 7c. Carbapenems 10a and 10b, containing minimal ethyl and ethylamine side chains, respectively, inactivated LdtMt1 with overall efficiencies 2.5- and 7.6-times lower than our reference compound (7a) containing a phenyl group linked to the carbapenem core by a triazole ring. Thus, the side chain of our compounds marginally contributed to the efficiency of the inactivation reaction. A similar conclusion has been previously reported for the analysis of the inactivation of the Enterococcus faecium L,D-transpeptidase by a limited series of compounds.32 The side chain of the azetidinone (β-lactam) ring was functionalized by esterification with acyl chlorides (Scheme 4). In comparison to the parental compound (10a), introduction of a phenyl (13a) or a pentafluorophenyl (13b) group led to a 2.8-fold decrease or a 2.3-fold increase in the acylation efficiency. Thus, bulky side chains were well tolerated on the F



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opportunistic pathogens and the selection of resistant bacteria. Optimization of carbapenems for the treatment of TB may therefore also include development of drugs that are only active against M. tuberculosis. Since Ldts are structurally unrelated to the classical D,D-transpeptidases38,39 and since Ldts are absent or unessential in most bacteria,17 targeting of Ldts appears to be an attractive approach to develop M. tuberculosis-specific inhibitors.

13b by BlaC were determined by spectrophotometry (Table 1). The catalytic efficiency of BlaC (kcat/Km) for the latter drugs ranged from 300 to 13,000 M−1 s−1. Three carbapenems, 7c, 7e, and 7f, which contained triazole rings and a phenyl or a butyl substituted by a carboxyl, were efficiently hydrolyzed by BlaC. In comparison to meropenem, the kcat/Km was increased 3.2− 13-fold due to large increases in the value of kcat (160−350 fold), which were only partially compensated by increases in the value of Km (21−61 fold). Three carbapenems, 7b, 10b, and 10c were hydrolyzed less efficiently by BlaC than meropenem (kcat/Km = ca. 350 versus 1000 M−1 s−1, respectively). These data indicate that functionalization of the side chains of carbapenems affords compounds that are superior or equal to clinically used carbapenems with respect to their stability toward hydrolysis by the β-lactamase BlaC of M. tuberculosis.

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EXPERIMENTAL SECTION

General Procedures. Solvents were dried using standard methods and distilled before use. Unless otherwise specified, materials were purchased from commercial suppliers and used without further purification. TLC: precoated silica gel thin-layer sheets 60 F254 (Merck) and detection by charring with 10% H2SO4 in ethanol followed by heating. Flash chromatography: silica gel 60 Å, 180−240 mesh from Merck. Spectra were recording using Bruker spectrometers AM250 or Bruker Advance II 500. Chemical shifts (δ) are expressed in ppm relative to residual CHCl3 (δ 7.26) or HDO (δ 4.79) for 1H, CDCl3 (δ 77.16) for 13C as internal references. Signals were assigned based on COSY and HSQC (13C). High-resolution mass spectroscopy (HRMS) was recorded using a Bruker micrOTOF spectrometer. Highperformance liquid chromatography (HPLC) was performed on a HPLC system with a reverse phase C-18 column (250 mm × 21 mm, Nucleosil, Macherey Nagel) using a solvent system consisting of CH3CN-H2O (linear gradient from 0:100 to 100:0 in 30 min) at a flow rate of 15 mL·min−1 and UV detection at 299 nm. The purity of all final compounds is ≥95%. The purity was determined by analytical HPLC. Acid but-3-ynyl Ester 1. Methanesulfonyl chloride (12 g, 106.5 mmol) and triethylamine (15 mL, 106.5 mmol) were added at 0 °C to a solution of 3-butyn-1-ol (5 g, 71 mmol) in DCM (90 mL). The reaction was stirred overnight at room temperature, then diluted with 2 M HCl and extracted with DCM. The combined extracts were washed with 1 M HCl, saturated NaHCO3 solution, brine, dried over MgSO4, filtered, and concentrated under reduce pressure to afford methanesulfonyl acid but-3-ynyl ester (10 g, 100%). 1H NMR (250 MHz, CDCl3): δ = 2.06 (t, J = 2.6 Hz, 1 H), 2.63−2.69 (td, J = 6.7, 2.7 Hz, 2 H), 3.05 (s, 3 H),, 4.31 (t, J = 6.8 Hz, 2 H). Methanesulfonyl acid but-3-ynyl ester (2 g, 13,4 mmol) in DMF (5 mL) was added dropwise at 0 °C to a stirring mixture of cesium carbonate (5.3 g, 16.2 mmol) and thioacetic acid (1.15 mL, 16.2 mmol) in DMF (10 mL), and the reaction was stirred 1 h at room temperature. The resulting solution was diluted in DCM and washed with brine. The combined organic layers were dried over MgSO4 and concentrated in vacuo. Distillation over reduced pressure gave 3-butynyl ethanethioate 1 (760 mg, 45%). 1H NMR (250 MHz, CDCl3): δ = 2.01 (t, J = 2.6 Hz, 1 H), 2.34 (s, 3 H), 2.44−2.51 (td, J = 6.7, 2.5 Hz, 2 H), 3.03 (t, J = 7.0 Hz, 2 H). Spectroscopic data were consistent with the literature data for this compound.40 Carbapenem Alkyne 4. To a solution of 3-butynyl ethanethioate 1 (290 mg, 2.26 mmol) in DMF (2 mL) was added a 1 M solution of NaOH (2.5 mL, 2.48 mmol). The reaction mixture was stirred 30 min at room temperature, then filtred on a DOWEX resin (50WX8-400). The resulting solution of 2 was cooled to 0 °C, and β-methyl vinyl phosphate 3 (0.74 mmol, 444 mg) and diisopropylethylamine (272 μL, 2.26 mmol) were successively added. The reaction mixture was stirred overnight at 4 °C. EtOAc was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/ EtOAc (1/9) as eluent to afford compound 4 (254 mg, 78%). 1H NMR (500 MHz, CDCl3): δ = 1.27 (d, J = 7.0 Hz, 3 H), 1.36 (d, J = 6.4 Hz, 3 H), 2.08 (t, J = 2.8 Hz, 1 H), 2.51−2.57 (m, 2 H), 2.91−2.97 (m, 2 H), 3.02−3.08 (m, 1 H), 3.26−3.28 (m, 1 H), 4.21−4.27 (m, 2 H), 5.23 (d, J = 13.1 Hz, 1 H), 5.51 (d, J = 13.1 Hz, 1 H), 7.65 (d, J = 8.7 Hz, 2 H), 8.21 (d, J = 8.7 Hz, 2 H).13C NMR (125 MHz, CDCl3): δ = 16.9, 20.3, 22.0, 30.2, 43.4, 56.1, 59.8, 65.4, 66.1, 70.8, 81.2, 123.8,

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CONCLUSION We have developed three routes of synthesis of carbapenems (Figure 1C), two of which for modification of the sulfur-linked side chain of the five-membered ring and one for the esterification of the hydroxyl group in the side chain of the β-lactam (azetidinone) ring. The overall efficiency of the acylation reaction (k2/Kapp) was 2.8−30 times greater for our carbapenems than for meropenem, which is currently evaluated for the treatment of tuberculosis.12 This was mainly due to higher values of k1 indicating that the first step of the acylation reaction, i.e., the formation of the oxyanion, is more rapid for our carbapenems. Of note, this step was previously shown to be determinant for the antibacterial activity of carbapenems.37 Overall the differences between carbapenems were moderate and may involve a negative impact of certain side chains, such as those present in meropenem and doripenem, which were not designed for optimal inhibition of L,D-transpeptidases. This premise is based on the observation that carbapenem 10a, with a minimal ethyl side chain, efficiently inactivated LdtMt1. The fact that modifications of the side chains only provided modest improvements to the efficiency of LdtMt1 inactivation is somewhat disappointing. However, it should be noted that inactivation by compounds containing the carbapenem core with minimal side chains (10a and 10b) is in itself very efficient. Moreover, most of the side chains introduced in our compounds were very well tolerated, indicating that other properties of carbapenems could be improved without reducing the efficiency of inactivation of the LdtMt1 target. Accordingly, we show that this approach can be used to reduce the hydrolysis of the drugs by the β-lactamase BlaC. For example, compound 7b was superior to meropenem both with respect to the efficiency of LdtMt1 acylation (20-fold increase in k2/Kapp) and reduced hydrolysis by BlaC (3.3-fold reduction in kcat/Km). Likewise, a 30-fold increase in k2/Kapp and a 2.8 reduction hydrolysis by BlaC were observed for carbapenem 10c. The versatile routes of synthesis of carbapenems described in this study will provide access to series of compounds for improvements of other properties of carbapenems. These properties may include development of an oral drug since meropenem requires frequent intravenous injection, and this mode of administration is a severe limitation for the treatment of tuberculosis (TB). Furthermore, carbapenems approved for use in humans are broad spectrum drugs. This was an asset in the context of their development, but the broad spectrum of approved carbapenems is a major issue in the context of TB since prolonged treatments with these drugs have adverse effects on the commensal flora, leading to infections by G



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Azide 5f. Sodium azide (6.2 g, 96.6 mmol) was added to a solution of 5-bromopentanoic acid (5 g, 27.6 mmol) in DMF (20 mL), and the reaction mixture was heated overnight at 85 °C. The solution was cooled to room temperature, EtOAc was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting 5-azidopentanoic acid (700 mg, 4.8 mmol) was dissolved in DCM (40 mL), and EDCI (1.4 g, 7.2 mmol) followed by HOBT (991 mg, 7.2 mmol) were added. The reaction mixture was allowed to stir for 30 min then cooled to 0 °C. 4-Nitrobenzoic acid was added, and the solution was stirred at room temperature for 24 h. DCM was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude was purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to give 5f (416 mg, 31%). 1H NMR (250 MHz, CDCl3): δ = 1.50−1.73 (m, 4 H), 2.38 (t, J = 6.8 Hz, 2 H), 3.23 (t, J = 6.8 Hz, 2 H), 5.14 (s, 2 H), 7.44 (d, J = 8.7 Hz, 2 H), 8.10 (d, J = 8.7 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 22.1, 28.3, 33.5, 51.0, 64.8, 123.8, 128.4, 143.3, 147.7, 172.6. HRMS: calcd for C12H13N4O4 [M − H]+: 277.0937; found: 277.0931. Azide 5h. Sodium azide (217 mg, 3.3 mmol) was added to a solution of 3-(2-bromoethyl)indole (500 mg, 2.2 mmol) in a H2O/ acetone 1/5 mixture (50 mL). The reaction mixture was stirred overnight at room temperature, EtOAc was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. Purification by flash chromatography using cyclohexane/EtOAc (8/ 2) as eluent gave compound 5h (341 mg, 82%). 1H NMR (250 MHz, CDCl3): δ = 3.08 (t, J = 7.1 Hz, 2 H), 3.58 (t, J = 7.1 Hz, 2 H), 7.08− 7.11 (m, 1 H), 7.13−7.26 (m, 2 H), 7.36−7.40 (m, 1 H), 7.58−7.61 (m, 1 H), 8.02 (bs, 1 H). 13C NMR (125 MHz, CDCl3): δ = 25.1, 51.7, 111.39, 112.4, 118.6, 119.6, 122.3, 127.2, 136.3. HRMS: calcd for C10H9N4 [M − H]+: 185.0827; found: 185.0819. General Procedure for Cu(I)-catalyzed 1,3 Cycloaddition. Conditions A: To a solution of compound 4 (1 equiv) in DMF were successively added azide 5 (1.2 equiv), sodium ascorbate (0.6 equiv., in water), and CuSO4 (0.3 equiv, in water). The heterogeneous mixture was stirred vigorously overnight at room temperature. EtOAc was then added, the phases were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/EtOAc as eluent to afford compounds 6. Conditions B: Caution: The reaction is exothermic and can be accompanied by dinitrogen release. It should not be performed in a closed vessel, and adequate cooling should always be available. Compound 4 (1 equiv) was added to a solution of copper(I) thiophene-2-carboxylate (CuTC, 0.3 equiv) in toluene. The reaction mixture was cooled to 0 °C, and azide 5 (1 equiv) was added. After stirring 8 h at room temperature, water and EtOAc were added, and the phases were separated. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/ EtOAc as eluent to afford compounds 6. Carbapenem 6a. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6a was obtained (45 mg, 55%) starting from compound 4 (60 mg, 0.14 mmol) and azide 5a (350 μL, 0.5 M in tert-butyl methyl ether, 0.17 mmol). 1H NMR (500 MHz, CDCl3): δ = 1.23 (d, J = 7.6 Hz, 3 H), 1.33 (d, J = 6.8 Hz, 3 H), 3.12−3.13 (m, 2 H), 3.16−3.20 (m, 1 H), 3.25−3.31 (m, 2 H), 3.42−3.49 (m, 1 H), 4.17−4.19 (m, 1 H), 4.21− 4.24 (m, 1 H), 5.16 (d, J = 13.5 Hz, 1 H), 5.45 (d, J = 13.5 Hz, 1 H), 7.41 (t, J = 7.2 Hz, 1 H), 7.48 (t, J = 7.7 Hz, 2 H), 7.60 (d, J = 8.5 Hz, 2 H), 7.67 (d, J = 7.9 Hz, 2 H), 7.81 (s, 1 H), 8.16 (d, J = 8.4 Hz, 2 H). 13 C NMR (125 MHz, CDCl3): δ = 16.7, 21.7, 26.3, 30.7, 43.1, 55.9, 59.6, 65.1, 65.7, 120.1, 120.4, 123.6, 123.8, 128.0, 128.8, 129.7, 136.8, 142.9, 145.4, 147.5, 152.1, 160.3, 172.7. HRMS: calcd for C27H27N5O6S [M + H]+: 550.1760; found: 550.1762.

128.3, 143.1, 147.7, 151.5, 160.4, 172.6. HRMS: calcd for C21H22N2O6SNa [M + Na]+: 453.1096; found: 453.1076. Azide 5b. To a solution of 2-azidobenzoic acid (5 mL, 0.25 M in tert-butyl methyl ether, 1.25 mmol) in DMF (5 mL) was added DMAP (76 mg, 0.625 mmol), followed by 4-nitrobenzyl alcohol (382 mg, 2.5 mmol). The resulting solution was cooled to 0 °C, and DCC (516 mg, 2.5 mmol) was added. The mixture was allowed to stir at room temperature for 24 h after which DCU precipitate was removed by filtration. The filtrate was evaporated in vacuo, and the crude was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to give 5b (103 mg, 27%). 1H NMR (250 MHz, CDCl3): δ = 5.44 (s, 2 H), 7.28−7.30 (m, 3 H), 7.71 (d, J = 8.6 Hz, 2 H), 8.21− 8.24 (m, 1 H), 8.37 (d, J = 8.6 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 65.7, 117.9, 124.0, 124.1, 125.8, 128.5, 128.6, 129.1, 143.4, 147.9, 152.6. Azide 5c. To a solution of methyl 3-azidobenzoate (3 mL, 0.5 M in tert-butyl methyl ether, 1.5 mmol) in a THF/MeOH 1/1 mixture (10 mL) was added a 1 M solution of NaOH (3 mL, 3 mmol), and the solution was stirred for 3 h at room temperature. The reaction mixture was quenched with 1 M HCl, and tert-butylmethyl ether was then added. The phases were separated, the aqueous layer was extracted with tert-butylmethyl ether, and the combined organic layers were dried over MgSO4. The resulting solution was diluted in DMF (2 mL), and DMAP (91 mg, 0.75 mmol) followed by 4-nitrobenzyl alcohol (459 mg, 3 mmol) were added. The reaction mixture was cooled to 0 °C, and DCC (619 mg, 3 mmol) was added. The mixture was allowed to stir at room temperature for 24 h after which DCU precipitate was removed by filtration. The filtrate was evaporated in vacuo, and the crude was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent affording 5c (103 mg, 23%). 1H NMR (250 MHz, CDCl3): δ = 5.45 (s, 2 H), 7.20−7.24 (m, 1 H), 7.44 (t, J = 7.8 Hz, 1 H), 7.6 (d, J = 8.7 Hz, 2 H), 7.69−7.71 (m, 1 H), 7.81−7.85 (m, 1 H), 8.23 (d, J = 8.7 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 65.6, 120.2, 123.9, 124.0, 126.2, 128.5, 130.1, 131.3, 140.9, 143.0, 147.9, 165.3. Azide 5d. To a solution of 4-azidobenzoic acid (4.5 mL, 0.2 M in tert-butyl methyl ether, 0.9 mmol) in DMF (5 mL) was added DMAP (11 mg, 0.09 mmol), followed by 4-nitrobenzyl alcohol (150 mg, 1 mmol). The resulting solution was cooled to 0 °C, and DCC (205 mg, 1 mmol) was added. The mixture was allowed to stir at room temperature for 24 h after which DCU precipitate was removed by filtration. The filtrate was evaporated in vacuo, and the crude was purified by flash chromatography using cyclohexane/EtOAc (9/1) as eluent to give 5d (61 mg, 23%). 1H NMR (250 MHz, CDCl3): δ = 5.44 (s, 2 H), 7.09 (d, J = 9.0 Hz, 2 H), 7.6 (d, J = 9.0 Hz, 2 H), 8.07 (d, J = 9.0 Hz, 2 H), 8.24 (d, J = 9.0 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 37.4, 91.1, 96.0, 98.0, 100.5, 103.7, 115.3, 117.5, 119.9, 137.4. Azide 5e. Sodium azide (170 mg, 2.61 mmol) was added to a solution of 4-(1-bromoethyl)benzoic acid (200 mg, 0.87 mmol) in a H2O/acetone 1/5 mixture (10 mL). The reaction mixture was stirred overnight at room temperature. EtOAc was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. Purification by flash chromatography using EtOAc as the eluent gave 4-(1-azidoethyl)benzoic acid (75 mg, 45%). 4-(1-Azidoethyl)benzoic acid (75 mg, 0.4 mmol) was then dissolved in DMF (1.5 mL), and DMAP (24 mg, 0.2 mmol) followed by 4-nitrobenzyl alcohol (120 mg, 0.8 mmol) were added. The reaction mixture was cooled to 0 °C, and DCC (161 mg, 0.8 mmol) was added. The mixture was allowed to stir at room temperature for 24 h after which DCU precipitate was removed by filtration. The filtrate was evaporated in vacuo, and the crude was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford 5e (73 mg, 57%). 1H NMR (250 MHz, CDCl3): δ = 1.54 (d, J = 6.8 Hz, 3 H), 4.65−4.73 (m, 1 H), 5.45 (s, 2 H), 7.43 (d, J = 8.7 Hz, 2 H), 7.60 (d, J = 8.7 Hz, 2 H), 8.09 (d, J = 8.2 Hz, 2 H), 8.24 (d, J = 8.2 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 21.7, 60.6, 65.3, 123.9, 126.6, 128.4, 129.3, 130.4, 143.3, 146.7, 147.9, 165.7. H



Article

Carbapenem 6b. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6b was obtained (43 mg, 43%) starting from compound 4 (60 mg, 0.14 mmol) and azide 5b (50 mg, 0.17 mmol). 1H NMR (500 MHz, CDCl3): δ = 1.25 (d, J = 6.9 Hz, 3 H), 1.34 (d, J = 6.2 Hz, 3 H), 3.15− 3.16 (m, 2 H), 3.19−3.24 (m, 1 H), 3.27 (dd, J = 6.7, 1.9 Hz, 1 H), 3.30−3.34 (m, 1 H), 3.43−3.49 (m, 1 H), 4.19−4.24 (m, 2 H), 5.17 (d, J = 14.1 Hz, 1 H), 5.24 (s, 2 H), 5.46 (d, J = 14.1 Hz, 1 H), 7.19 (t, J = 7.4 Hz, 1 H), 7.29 (d, J = 7.4 HZ, 1 H), 7.45 (t, J = 7.7 Hz, 1 H), 7.52 (d, J = 8.1 Hz, 2 H), 7.62 (d, J = 8.8 Hz, 2 H), 8.16−8.20 (m, 4 H). 13C NMR (125 MHz, CDCl3): δ = 16.9, 21.9, 26.3, 30.7, 43.3, 56.1, 59.9, 65.3, 65.8, 65.9, 123.2, 123.8, 123.9, 124.2, 124.3, 128.2, 128.5, 130.1, 143.0, 143.2, 147.7, 147.8, 151.7, 153.1, 160.5, 172.9. Carbapenem 6c. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6c was obtained (56 mg, 60%) starting from compound 4 (55 mg, 0.13 mmol) and azide 6c (41 mg, 0.15 mmol). 1H NMR (500 MHz, CDCl3):δ = 1.23 (d, J = 6.9 Hz, 3 H), 1.33 (d, J = 6.1 Hz, 3 H), 3.11− 3.21 (m, 3 H), 3.26 (dd, J = 7.1, 1.9 Hz, 1 H), 3.29−3.36 (m, 1 H), 3.45−3.48 (m, 1 H), 4.19−4.25 (m, 2 H), 5.15 (d, J = 13.8 Hz, 1 H), 5.44 (d, J = 13.8 Hz, 1 H), 5.47 (s, 2 H), 7.57−7.63 (m, 5 H), 7.96− 7.97 (m, 1 H), 8.11 (d, J = 7.9 Hz, 1 H), 8.14 (d, J = 8.7 Hz, 2 H), 8.23 (d, J = 8.7 Hz, 2 H), 8.35 (s, 1 H).13C NMR (125 MHz, CDCl3): δ = 16.9, 21.9, 26.4, 30.8, 43.2, 56.0, 59.8, 65.2, 65.8, 65.9, 121.4, 123.7, 124.0, 125.1, 128.1, 128.7, 129.8, 130.3, 131.4, 137.3, 142.7, 143.1, 147.6, 147.9, 152.0, 160.5, 164.9, 172.9. HRMS: calcd for C35H33N6O6S [M + H]+: 729.1979; found: 729.1967. Carbapenem 6d. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6d was obtained (28 mg, 56%) starting from compound 4 (30 mg, 0.07 mmol) and azide 5d (25 mg, 0.08 mmol). 1H NMR (500 MHz, CDCl3): δ = 1.24 (d, J = 6.9 Hz, 3 H), 1.34 (d, J = 6.4 Hz, 3 H), 3.13− 3.16 (m, 2 H), 3.18−3.24 (m, 1 H), 3.27 (dd, J = 6.5, 1.9 Hz, 1 H), 3.29−3.38 (m, 1 H), 3.43−3.50 (m, 1 H), 4.2 (dd, J = 8.7, 2.0 Hz, 1 H), 4.21−4.26 (m, 1 H), 5.17 (d, J = 13.2 Hz, 1 H), 5.45 (d, J = 13.2 Hz, 1 H), 5.47 (s, 1 H), 7.61 (t, J = 9.2 Hz, 4 H), 7.82 (d, J = 8.1 Hz, 2 H), 7.91 (s, 1 H), 8.16 (d, J = 8.1 Hz, 2 H), 8.21 (d, J = 8.1 Hz, 2 H), 8.26 (d, J = 8.8 Hz, 2 H).13C NMR (125 MHz, CDCl3): δ = 16.9, 21.9, 26.4, 30.8, 43.2, 56.1, 59.8, 65.3, 65.7, 65.9, 120.0, 123.8, 124.0, 127.1, 128.2, 128.6, 129.7, 131.6, 140.4, 143.0, 146.0, 147.7, 148.0, 151.9, 160.5, 164.9, 172.8. HRMS: calcd for C35H33N6O10S [M + H]+: 729.1979; found: 729.1968. Carbapenem 6e. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6e was obtained (69 mg, 56%) starting from compound 4 (70 mg, 0.16 mmol) and azide 5e (80 mg, 0.24 mmol).1H NMR (500 MHz, CDCl3): δ = 1.18 (d, J = 6.9 Hz, 3 H), 1.29−1.32 (m, 3 H), 1,95 (t, J = 6.2 Hz, 3 H), 2.96−3.03 (m, 2 H), 3.07−3.11 (m, 1 H), 3.20−3.24 (m, 2 H), 3.39−3.42 (m, 1 H), 4.08−4.13 (m, 1 H), 4.18−4.22 (m, 1 H), 5.17 (d, J = 14.0 Hz, 1 H), 5.41 (s, 1 H), 5.45 (d, J = 14.0 Hz, 1 H), 5.80 (quint, J = 6.3 Hz, 1 H), 7.27−7.29 (m, 2 H), 7.34 (s, 1 H), 7.56 (d, J = 7.9 Hz, 2 H), 7.61 (d, J = 8.5 Hz, 2 H), 8.03 (d, J = 7.8 Hz, 2 H), 8.14 (d, J = 8.7 Hz, 2 H), 8.20 (d, J = 8.7 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 16.6, 21.2, 21.8, 26.5, 30.8, 43.2, 56.0, 59.7, 59.9, 65.2, 65.4, 65.8, 120.9, 123.7, 123.9, 126.6, 128.2, 128.4, 129.7, 130.5, 143.1, 143.2, 144.9, 145.4, 145.5, 147.6, 147.8, 152.4, 165.4, 172.9. HRMS: calcd for C37H37N6O10S [M + H]+: 757.2292; found: 757.2277. Carbapenem 6f. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6f was obtained (20 mg, 41%) starting from compound 4 (30 mg, 0.07 mmol) and azide 5f (23 mg, 0.08 mmol). 1H NMR (500 MHz, CDCl3): δ = 1.21 (d, J = 7.0 Hz, 3 H), 1.34 (d, J = 6.1 Hz, 3 H), 1.63− 1.69 (m, 2 H), 1.90−1.96 (m, 2 H), 2.42−2.45 (m, 2 H), 3.01−3.06 (m, 2 H), 3.10−3.14 (m, 1 H), 3.24−3.25 (m, 2 H), 3.42−3.45 (m, 1 H), 4.16 (dd, J = 9.2, 1.6 Hz, 1 H), 4.23 (quint, J = 4.6 Hz, 1 H), 4.30−4.33 (m, 2 H), 5.18 (s, 1 H), 7.49 (d, J = 8.5 Hz, 2 H), 7.65 (d, J = 8.5 Hz, 2 H), 8.19−8.21 (m, 4 H). 13C NMR (125 MHz, CDCl3): δ = 16.9, 21.8, 21.9, 26.5, 29.6, 31.0, 33.2, 43.3, 50.0, 56.1, 59.8, 65.0, 65.3, 65.9, 122.0, 123.8, 123.9, 128.3, 128.5, 143.1, 144.8, 147.7, 147.8,

152.5, 160.5, 172.5, 172.9. HRMS: calcd for C33H36N6O10SNa [M + Na]+: 731.2112; found: 731.2107. Carbapenem 6g. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions B), carbapenem 6g was obtained (17 mg, 37%) starting from compound 4 (30 mg, 0.07 mmol) and azide 5g (17 mg, 0.07 mmol). 1H NMR (500 MHz, CDCl3): δ = 1.12 (d, J = 7.4 Hz, 3 H), 1.32 (d, J = 6.1 Hz, 3 H), 2.18 (s, 3 H), 2.98−3.04 (m, 1 H), 3.06−3.13 (m, 2 H), 3.17−3.21 (m, 2 H), 3.24−3.31 (m, 1 H), 399 (dd, J = 8.9, 2.6 Hz, 1 H), 4.20−4.23 (m, 1 H), 5.25 (d, J = 13.5 Hz, 1 H), 5.48 (d, J = 13.5 Hz, 1 H), 7.64 (d, J = 8.2 Hz, 2 H), 7.72 (d, J = 8.9 Hz, 2 H), 7.95−7.97 (m, 2 H), 8.02 (s, 1 H), 8.20 (d, J = 8.7 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 16.6, 21.8, 24.7, 26.3, 30.3, 43.0, 55.6, 59.7, 65.3, 65.7, 119.7, 121.8, 123.7, 123.8, 128.2, 129.5, 130.1, 142.9, 144.5, 144.9, 147.6, 151.4, 160.3, 169.1, 172.7. HRMS: calcd for C29H31N6O9S2 [M + H]+: 671.1594; found: 671.1583. Carbapenem 6h. Following the general procedure for Cu(I)catalyzed 1,3 cycloaddition (Conditions A), carbapenem 6h was obtained (60 mg, 71%) starting from compound 4 (60 mg, 0.14 mmol) and azide 5h (31 mg, 0.17 mmol). 1H NMR (500 MHz, CDCl3): δ = 1.11 (d, J = 6.9 Hz, 3 H), 1.27 (d, J = 3 H), 2.84−2.88 (m, 2 H), 2.99−3.04 (m, 1 H), 3.08−3.12 (m, 1 H), 3.17−3.22 (m, 3 H), 3.30−3.3 (m, 1 H), 4.10 (d, J = 8.2 Hz, 1 H), 4.15 (quint, J = 6.5 Hz, 1 H), 4.46−4.52 (m, 2 H), 5.10 (d, J = 13.8 Hz, 1 H), 5.37 (d, J = 13.8 Hz, 1 H), 6.85 (s, 1 H), 7.01 (t, J = 7.5 Hz, 1 H), 7.08 (d, J = 7.5 Hz, 1 H), 7.25 (d, J = 8.3 Hz, 1 H), 7.40 (d, J = 7.7 Hz, 1 H), 7.50 (d, J = 8.2 Hz, 2 H), 8.04 (s, 1 H). 13C NMR (125 MHz, CDCl3): δ = 16.9, 21.9, 26.0, 26.6, 30.9, 43.3, 50.9, 56.2, 59.7, 65.3, 65.9, 110.5, 111.6, 118.1, 119.5, 122.1, 123.2, 123.3, 123.7, 126.7, 128.1, 136.3, 143.0, 147.8, 153.5, 160.8, 173.0. General Procedure for the Deprotection of para-Nitrobenzyl (PNB) Group by Hydrogenation. A pressure reactor was charged with para-nitrobenzyl protected carbapenem (1 equiv), 10 wt % Pd/C (3 equiv), and a 1/1 mixture of THF/triethylammonium bicarbonate buffer (pH 8.5). The reactor was pressurized with 3.5 bar H2, and the closed system was stirred for 2 h. Palladium was removed by filtration through Celite, and the filtrate concentrated. The residue was purified by HPLC. The appropriate fractions were collected and lyophilized, to give triethylammonium salt. Carbapenem 7a. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7a was obtained (8.5 mg, 27%) starting from compound 6a (42 mg, 0.076 mmol). 1H NMR (250 MHz, D2O): δ = 1.05 (d, J = 6.8 Hz, 3 H), 1.17 (d, J = 6.8 Hz, 3 H), 1.24 (t, J = 7.4 Hz, 9 H), 3.00−3.09 (m, 3 H), 3.16 (q, J = 7.6 Hz, 6 H), 3.24−3.28 (m, 1 H), 3.69−3.73 (m, 1 H), 4.01−4.11 (m, 1 H), 7.51−7.61 (m, 3 H), 7.66−7.69 (m, 2 H), 8.21 (s, 1 H). HRMS: calcd for C20H23N4O4S [M + H]+: 415.1440; found: 415.1444. Carbapenem 7b. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7b was obtained (4.3 mg, 13%) starting from compound 6b (43 mg, 0.059 mmol). 1H NMR (250 MHz, D2O): δ = 1.16 (d, J = 7.1 Hz, 3 H), 1.24−1.33 (m, 12 H), 3.08−3.31 (m, 10 H), 3.35−3.38 (m, 2 H), 3.91−3.95 (m, 1 H), 4.17− 4.22 (m, 1 H), 6.98−7.11 (m, 2 H), 7.33 (d, J = 8.0 Hz, 1 H), 7.43 (t, J = 8.0 Hz, 1 H), 8.07 (s, 1 H). HRMS: calcd for C21H21N4O6S [M − H]+: 457.1182; found: 457.1195. Carbapenem 7c. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7c was obtained starting from compound 6c (56 mg, 0.076 mmol). 1H NMR (250 MHz, D2O): δ = 1.14 (d, J = 7.3 Hz, 3 H), 1.23−1.32 (m, 21 H), 3.17−3.25 (m, 15 H), 3.33−3.36 (m, 2 H), 3.53−3.61 (m, 1 H), 3.85−3.98 (m, 1 H), 4.13− 4.17 (m, 1 H), 7.69 (t, J = 7.8 Hz, 1 H), 7.88 (d, J = 8.7 Hz, 1 H), 8.03 (d, J = 8.7 Hz, 1 H), 8.21 (s, 1 H), 8.34 (s, 1 H). HRMS: calcd for C21H23N4O6S [M + H]+: 459.1338; found: 459.1336. Carbapenem 7d. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7d was obtained (2.4 mg, 21%) starting from compound 6d (13 mg, 0.017 mmol). 1H NMR (250 MHz, D2O): δ = 1.08 (d, J = 6.6 Hz, 3 H), 1.19 (d, J = 6.6 Hz, 3 H), 1.28 (t, J = 7.0 Hz, 18 H), 3.04−3.23 (m, 4 H), 3.25−3.33 (m, 2 H), 3.43 (q, J = 6.6 Hz, 12 H), 3.77−3.79 (m, 1 H), 4.07−4.13 (m, 1 H), 7.78 (d, J = 8.2 Hz, 2 H), 8.03 (d, J = 8.2 Hz, 2 H), 8.31 (s, 1 H). I



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HRMS: calcd for C21H21N4O6S [M − H]+: 457.1187; found: 457.1191. Carbapenem 7e. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7e was obtained (9.1 mg, 18%) starting from compound 6e (66 mg, 0.08 mmol). 1HNMR (250 MHz, D2O): δ = 1.06 (d, J = 7.3 Hz, 3 H), 1.25−1.31 (m, 21 H), 1.97 (t, J = 7.7 Hz, 3 H), 3.00−3.12 (m, 3 H), 3.16−3.24 (m, 12 H), 3.39−3.32 (m, 1 H), 3.48−3.55 (m, 1 H), 3.71−3.75 (m, 2 H), 5.92−5.98 (m, 1H), 7.39−7.42 (m, 2 H), 7.89−7.95 (m, 3 H). HRMS: calcd for C23H25N4O6S [M − H]+: 485.1495; found: 485.1473. Carbapenem 7f. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7f was obtained (6.4 mg, 10%) starting from compound 6f (69 mg, 0.097 mmol). 1H NMR (250 MHz, D2O): δ = 1.10 (d, J = 7.5 Hz, 3 H), 1.25−1.13 (m, 21 H), 1.50−1.56 (m, 2 H), 1.87−1.93 (m, 3 H), 2.25−2.30 (m, 3 H), 2.99− 3.08 (m, 2 H), 3.20 (q, J = 6.8 Hz, 12 H), 3.90−3.93 (m, 1 H), 4.19− 4.23 (m, 1 H), 4.37−4.42 (m, 3 H), 7.82 (s, 1 H). HRMS: calcd for C19H27N4O6S [M + H]+: 439.1651; found: 439.1649. Carbapenem 7g. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 7g was obtained (5.4 mg, 17%) starting from compound 6g (34 mg, 0.051 mmol). 1H NMR (250 MHz, D2O): δ = 1.10 (d, J = 6.8 Hz, 3 H), 1.25−1.30 (m, 12 H), 2.19 (s, 3 H), 3.02−3.10 (m, 3 H), 3.15−3.21 (m, 6 H), 3.30−3.37 (m, 3 H), 3.91−3.94 (m, 1 H), 4.17−4.24 (m, 1 H), 7.56−7.60 (m, 2 H), 7.77−7.81 (m, 3 H). Compound 7h. Following the general procedure for deprotection of PNB by hydrogenation, compound 7h was obtained (14 mg, 26%) starting from compound 6h (60 mg, 0.097 mmol). 1H NMR (250 MHz, D2O): δ = 1.03 (d, J = 7.1 Hz, 3 H), 1.24−1.29 (m, 12 H), 2.74−2.92 (m, 4 H), 3.12−3.23 (m, 7 H), 3.29−3.39 (m, 3 H), 3.94− 3.98 (m, 1 H), 4.16−4.21 (m, 1 H), 4.62−4.68 (m, 2 H), 7.01−7.09 (m, 2 H), 7.18 (t, J = 7.0 Hz, 1 H), 7.32−7.35 (m, 2 H), 7.44 (d, J = 7.7 Hz, 1 H). HRMS: calcd for C24H27N5O4S [M + H]+: 482.1862; found: 482.1852. Thiol 8b. A solution of cysteamine (3.9 mmol, 300 mg) in MeCN (10 mL) was cooled to 0 °C, and diisopropylethylamine (1.6 mL, 8.9 mmol) was added. After stirring for 5 min, chlorotrimethylsilane (641 μL, 5.0 mmol) was added, and the reaction mixture was stirred for 15 min at 0 °C. para-Nitrobenzyl chloroformate (838 mg, 3.9 mmol) and diisopropylethylamine (700 μL, 3.9 mmol) were then added at this temperature, and the mixture stirred for 1 h 30 min. The reaction was quenched with water (2 mL) and warmed to room temperature. DCM was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/EtOAc (5/5) as the eluent to afford compound 12a as a yellow solid (645 mg, 66%). 1H NMR (250 MHz, D2O): δ = 1.36 (d, J = 8.1 Hz, 1 H), 2.64−2.73 (m, 2 H), 3.40 (q, J = 6.3 Hz, 2 H), 5.20 (s, 2 H), 7.51 (d, J = 8.0 Hz, 2 H), 8.22 (d, J = 8.0 Hz, 2 H). Carbapenem 9b. A solution of β-methyl vinyl phosphate 3 (0.16 mmol, 100 mg) in MeCN (2 mL) was cooled to 0 °C, and diisopropylethylamine (58 μL, 0.32 mmol) followed by 4-nitrobenzyl (2-mercaptoethyl)carbamate8b (52 mg, 0.19 mmol) were added. The reaction mixture was stirred overnight at 4 °C. EtOAc was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/EtOAc (2/8) as the eluent to afford compound 9b as a pale solid (75 mg, 74%). 1H NMR (500 MHz, CDCl3): δ = 1.25 (d, J = 6.7 Hz, 3 H), 1.37 (d, J = 6.7 Hz, 3 H), 3.08−3.13 (m, 1 H), 3.24−3.28 (m, 1 H), 3.36−3.42 (m, 1 H), 3.45− 3.52 (m, 2 H), 4.21−4.27 (m, 2 H), 5.19 (s, 2 H), 5.23 (d, J = 14.1 Hz, 1 H), 5.51 (d, J = 14.1 Hz, 1 H), 7.50 (d, J = 8.7 Hz, 2 H), 7.66 (d, J = 8.7 Hz, 2 H), 8.22 (d, J = 8.7 Hz, 4 H). HRMS: calcd for C27H27N4O10S [M − H]+: 599.1448; found: 599.1466. Carbapenem 9c. A solution of β-methyl vinyl phosphate 3 (0.33 mmol, 200 mg) in MeCN (5 mL) was cooled to 0 °C, and diisopropylethylamine (117 μL, 0.66 mmol) followed by 2-phenylethanethiol 8c (90 μL, 0.66 mmol) were added. The reaction mixture

was stirred overnight at 4 °C. EtOAc was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/EtOAc (3/7) as the eluent to afford compound 9c as a pale solid (135 mg, 83%). 1H NMR (500 MHz, CDCl3): δ = 1.12 (d, J = 7.7 Hz, 3 H), 1.24 (d, J = 6.2 Hz, 3 H), 2.82−2.86 (m, 2 H), 2.94− 3.05 (m, 2 H), 3.16 (dd, J = 6.8, 2.5, 1 H), 3.19−3.25 (m, 1 H), 4.03− 4.05 (m, 1 H), 4.13 (quint, J = 6.3 Hz, 1 H), 5.13 (d, J = 13.5 Hz, 1 H), 5.40 (d, J = 13.5 Hz, 1 H), 7.09−7.11 (m, 2 H), 7.14−7.16 (m, 1 H), 7.19−7.22 (m, 2 H), 7.54 (d, J = 8.5 Hz, 2 H), 8.09 (d, J = 8.5 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 16.8, 21.8, 32.9, 36.2, 43.3, 56.0, 59.5, 65.2, 65.8, 123.4, 123.7, 126.8, 128.1, 128.5, 128.6, 139.1, 143.1, 147.5, 152.8, 160.4, 172.9. HRMS: calcd for C25H27N2O6S [M + H]+: 483.1590; found: 483.1574. Carbapenem 10b. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 10b was obtained (19 mg, 40%) starting from compound 9b (100 mg, 0.16 mmol). 1H NMR (500 MHz, D2O): δ = 1.25 (d, J = 7.4 Hz, 3 H), 1.34 (d, J = 6.6 Hz, 3 H), 2.95−3.01 (m, 1 H), 3.16−3.21 (m, 1 H), 3.25−3.30 (m, 1 H), 3.32−3.37 (m, 1 H), 3.42−3.46 (m, 1 H), 3.50−3.51 (m, 1 H), 4.25− 4.30 (m, 2 H).MS: calcd for C11H17N2O4S [M + H]+: 287.1066; found: 287.1054. Carbapenem 10c. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 10c was obtained (24 mg, 20%) starting from compound 9c (135 mg, 0.30 mmol). 1H NMR (250 MHz, D2O): δ = 1.08 (d, J = 7.0 Hz, 3 H), 1.24−1.30 (m, 12 H), 2.98−3.03 (m, 3 H), 3.14−3.23 (m, 7 H), 3.30−3.33 (m, 2 H), 3.84− 3.88 (m, 1 H), 4.16−4.21 (m, 1 H), 7.32−7.36 (m, 5 H). HRMS: calcd for C18H22NO4S [M + H]+: 348.1270; found: 348.1260. Carbapenem 12a. A solution of carbapenem 9a (0.10 mmol, 41 mg) in DCM (2 mL) was cooled to 0 °C, and triethylamine (28 μL, 0.20 mmol), DMAP (24 mg, 0.20 mmol), and benzyl chloride 11a (23 μL, 0.20 mmol) were added. The reaction mixture was warmed to room temperature and stirred overnight. DCM was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/EtOAc (7/3) as the eluent to afford compound 12a as a pale solid (32 mg, 63%). 1H NMR (500 MHz, CDCl3): δ = 1.28 (d, J = 7.3 Hz, 3 H), 1.32 (t, J = 7.3 Hz, 3 H), 1.55 (d, J = 6.4 Hz, 3 H), 2.79−2.91 (m, 2 H), 3.36−3.43 (m, 1 H), 3.54 (dd, J = 2.5, 7.7, 1 H), 4.28 (dd, J = 2.4, 9.3 Hz, 1 H), 5.26 (d, J = 13.9 Hz, 1 H), 5.47 (d, J = 13.9 Hz, 1 H), 5.54 (quint, J = 6.6 Hz, 1 H), 7.42 (t, J = 7.9 Hz, 2 H), 7.54−7.57 (m, 1 H), 7.63 (d, J = 7.9 Hz, 2 H), 8.02 (d, J = 7.9 Hz, 2 H), 8.17 (d, J = 7.9 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 13.8, 16.0, 17.7, 24.8, 42.2, 56.1, 56.4, 64.2, 68.3, 122.3, 122.8, 127.2, 127.5, 128.7, 129.0, 132.3, 142.1, 146.7, 152.2, 159.4, 164.7, 169.8. HRMS: calcd for C26H27N2O7S [M + H]+: 511.1539; found: 511.1539. Carbapenem 12b. A solution of carbapenem 9a (0.15 mmol, 61 mg) in DCM (3 mL) was cooled to 0 °C, and triethylamine (45 μL, 0.30 mmol), DMAP (40 mg, 0.30 mmol), and pentafluorobenzyl chloride 11b (47 μL, 0.30 mmol) were added. The reaction mixture was warmed to room temperature and stirred overnight. DCM was then added, and the organic layer was washed with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography using cyclohexane/EtOAc (7/3) as the eluent to afford compound 12b as a pale solid (73 mg, 81%). 1H NMR (500 MHz, CDCl3): δ = 1.28 (d, J = 7.5 Hz, 3 H), 1.33 (t, J = 7.2 Hz, 3 H), 1.57 (d, J = 6.3 Hz, 3 H), 2.82−2.92 (m, 2 H), 3.39 (quint, J = 8.2 Hz, 1 H), 3.50 (dd, J = 2.2, 8.4 Hz, 1 H), 4.21 (dd, J = 2.3, 6.6 Hz, 1 H), 5.25 (d, J = 13.6 Hz, 1 H), 5.48 (d, J = 13.6 Hz, 1 H), 5.54−5.59 (m, 1 H), 7.65 (d, J = 8.4 Hz, 2 H), 8.21 (d, J = 8.4 Hz, 2 H). 13C NMR (125 MHz, CDCl3): δ = 14.7, 16.9, 18.7, 25.8, 43.2, 56.9, 57.2, 65.3, 72.1, 123.0, 123.8, 128.3, 143.1, 147.7, 153.6, 160.3, 169.8. 19F NMR (471 MHz, CDCl3): −159.8 (m, 2 F), −147.7 (m, 1 F), −138.4 (m, 2 F). HRMS: calcd for C26H22F5N2O7S [M + H]+: 601.1068; found: 601.1058 J



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Carbapenem 13a. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 13a was obtained (7.0 mg, 10%) starting from compound 12a (33 mg, 0.06 mmol). 1H NMR (250 MHz, D2O): δ = 1.17−1.26 (m, 12 H), 1.45 (d, J = 6.7 Hz, 3 H), 2.70−2.89 (m, 2 H), 3.16 (q, J = 7.1 Hz, 6 H), 3.30−3.44 (m, 1 H), 3.71−3.74 (m, 1 H), 4.31−4.36 (m, 1 H), 5.42−5.55 (m, 1 H), 7.42− 7.53 (m, 2 H), 7.62−7.68 (m, 1 H), 8.03 (d, J = 7.4 Hz, 2 H). HRMS: calcd for C19H22NO5S [M + H]+: 376.1219; found: 376.1230. Carbapenem 13b. Following the general procedure for deprotection of PNB by hydrogenation, carbapenem 13b was obtained (7.3 mg, 10%) starting from compound 12b (113 mg, 0.19 mmol). 1H NMR (250 MHz, D2O): δ = 1.29−1.33 (m, 12 H), 1.54 (d, J = 6.1 Hz, 3 H), 2.74−2.97 (m, 2 H), 3.23 (d, J = 7.4 Hz, 6 H), 3.43−3.79 (m, 1 H), 3.76−3.79 (m, 1 H), 4.31−4.36 (m, 1 H), 5.61−5.66 (m, 1 H). HRMS: calcd for C19H16F5N2O5S [M − H]−: 464.0591; found: 464.0605. Determination of Kinetic Parameters for Inactivation of the L,D-Transpeptidase LdtMt1 by β-Lactams. LdtMt1 was produced in Escherichia coli and purified from a clarified lysate by affinity chromatography on Ni2+-nitrilotriacetate-agarose resin (Qiagen GmbH) and by size-exclusion chromatography (Superdex 75 HL26/ 60 column, Amersham Pharmacia Biotech) in 100 mM sodiumphosphate buffer (pH 6.4) containing 300 mM NaCl.34 Kinetics of the inactivation of LdtMt1 by carbapenems were determined by stoppedflow spectrofluorimetry in 100 mM sodium-phosphate (pH 6.0) at 10 °C.34 The kinetic parameters k1, k2, and k2/Kapp were calculated, as previously described.34,37 Determination of BlaC Kinetic Parameters for Hydrolysis of Carbapenems. The β-lactamase BlaC was produced in E. coli and purified by affinity (Ni-NTA resin, Sigma-Aldrich) and size exclusion (Superdex 200 HL26/60 column, Amersham Pharmacia Biotech) chromatographies, as previously described.41 Hydrolysis of β-lactams was determined by spectrophotometry in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.4) at 20 °C. For carbapenems that were hydrolyzed by BlaC with Km values higher than 10 μM, kinetic parameters kcat and Km were determined as described by Soroka et al.41 For other compounds, kcat and Km were obtained using nitrocefin in the competition assay described by Tremblay et al.42

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ABBREVIATION USED BlaC, beta-lactamase of Mycobacterium tuberculosis; LdtMt1, L,Dtranspeptidase of Mycobacterium tuberculosis; DR-TB, drugresistant TB; GlcNAc, N-acetylglucosaminyl; MurNGlyc, Nglycolylmuramyl; DIPEA, N,N-diisopropylethylamine; DAP, mesodiaminopimelic acid; Ldts, L,D-transpeptidases; CuAAC, copper(I)-catalyzed 1,3 cycloaddition; PNB, para-nitrobenzyl; CuTC, CuI thiophene carboxylate; MES, 2-(N-morpholino)ethanesulfonic acid

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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00096. NMR and mass spectra (PDF) Compound data (CSV)

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*E-mail: [email protected]. Phone (33)-1-44-27-5455. *E-mail: [email protected]. Phone: (33)-1-42-86-40-26. Author Contributions #

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by grants from the European Commission Seventh Framework Program ORCHID project No. 261378 and from the Agence National de la Recherche (ANR), Project CARBATUB (N° ANR 2011 BSV5 024 01). K



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Routes of Synthesis of Carbapenems for Optimizing Both the

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